Mutations associated with cystic fibrosis

ABSTRACT

The present invention provides novel mutations identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that can be used for a more accurate diagnosis of cystic fibrosis (CF) and CF related disorders. Methods for testing a sample obtained from a subject to determine the presence of one or more mutations in the CFTR gene are provided wherein the presence of one or more mutations indicates that the subject has CF or a CF related disorder, or is a carrier of a CFTR mutation.

The present application is a continuation application of pending U.S.patent application Ser. No. 14/976,790, filed Dec. 21, 2015, entitled“Mutations Associated With Cystic Fibrosis,” which is a continuationapplication of U.S. patent application Ser. No. 14/271,106, filed May 6,2014, entitled “Mutations Associated With Cystic Fibrosis,” now U.S.Pat. No. 9,234,243, which is a continuation application of U.S. patentapplication Ser. No. 13/053,626, filed Mar. 22, 2011, entitled“Mutations Associated With Cystic Fibrosis,” now U.S. Pat. No.8,728,731, which claimed priority under 35 USC 119(e) from U.S.Provisional Patent Application No. 61/316,321 filed Mar. 22, 2010 andU.S. Provisional Patent Application No. 61/359,029 filed Jun. 28, 2010.The disclosures of U.S. Provisional Patent Application Nos. 61/316,321and 61/359,029, and U.S. patent application Ser. Nos. 13/053,626,14,271,106 and 14/976,790 are incorporated by reference in theirentireties herein.

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF) is the most common severe autosomal recessivegenetic disorder in the Caucasian population. It affects approximately 1in 2,500 Caucasian live births in North America (Boat et al, TheMetabolic Basis of Inherited Disease, 6th ed, pp 2649-2680, McGraw Hill,NY (1989)). The incidence of disease is lower in African American,Hispanic and Asian individuals. Approximately 1 in 25 Caucasian personsare carriers of the disease. The responsible gene has been localized toa 250,000 base pair genomic sequence present on the long arm ofchromosome 7. This sequence encodes a membrane-associated protein calledthe “cystic fibrosis transmembrane regulator” (or “CFTR”). The CFTR genecontains 27 exons and encodes a protein of 1480 amino acids. Severalregions are contemplated to have functional importance in the CFTRprotein, including two areas for ATP binding, termed Nucleotide BindingFolds (NBF), a Regulatory (R) region that has multiple potential sitesfor phosphorylation by protein kinases A and C, and two hydrophobicregions believed to interact with cell membranes.

The major symptoms of classical cystic fibrosis include chronicpulmonary disease, pancreatic exocrine insufficiency, congenital absenceof the vas deferens in males and elevated sweat electrolyte levels. Thesymptoms are consistent with CF being an exocrine disorder. Althoughrecent advances have been made in the analysis of ion transport acrossthe apical membrane of the epithelium of CF patient cells, it is notclear that the abnormal regulation of chloride channels represents theonly defect in the disease. Mutations in the CFTR gene are alsoassociated with atypical CF and monosymptomatic diseases such ascongential absence of the vas deferens in males, idiopathic chronicpancreatitis and chronic sinusitis (Noone and Knowles, Respir. Res.,vol. 2, p. 328 (2001); Southern, Respiration, vol. 74, p. 241 (2007)). Avariety of CFTR gene mutations are known. One of them leads to theomission of phenylalanine residue 508 within the first putative NBFdomain. This mutation, termed ΔF508, accounts for about 70% of the CFTRchromosomes in Caucasian patients and was highly associated with thepredominant haplotype found on chromosomes of Caucasian CF patients(Kerem, et al., Science, vol. 245, p. 1073 (1989); Lemna, et al., NewEngl. J. Med., vol. 322, p. 291 (1990)). However, the haplotypesassociated with Caucasian CF chromosomes without ΔF508 also existalthough less common, confirming that allelic heterogeneity is presentin CF and CF related disorders.

Therefore, there is a need for more effective genetic screening forother CFTR mutant alleles which are present in the other 30% ofCaucasian CF patients, as well as other alleles found in other racialand ethnic groups. Knowledge of such alleles can be used to designprobes for screening and/or testing, as well as to devise otherscreening and/or testing methods. The more complete the set of probesavailable for CFTR mutant alleles, the more accurate the diagnoses.

SUMMARY OF THE INVENTION

The present invention provides methods, products and systems relating tonovel mutations identified in the CFTR gene that can be used for moreaccurate diagnosis of CF and CF related disorders.

In one aspect, the present invention provides a method for testing formutations in the CFTR gene, which comprises testing a sample obtainedfrom a subject to determine the presence of one or more mutationsselected from Table 1, 2, 3, or 4 in the CFTR gene or protein, whereinthe presence of the one or more mutations indicates that the subject hasCF or a CFTR related disorder, is at risk of developing CF or a CFrelated disorder, or is a carrier of a CFTR mutation. In someembodiments, the one or more mutations are selected from Table 1, 2 or3. In some embodiments, the one or more mutations are selected fromTable 1 or 2. In some embodiments, the one or more mutations areselected from Table 1. In some embodiments, the one or more mutationsselected from Table 1, 2, 3, or 4 are part of a panel of CFTR mutations.

Yet other embodiments of the present invention comprise systems forperforming the method. For example, the system may comprise a station ordevice for testing a sample obtained from a subject to determine thepresence of one or more mutations selected from Table 1, 2, 3, or 4 inthe CFTR gene or protein, wherein the presence of the one or moremutations indicates that the subject has CF or a CFTR related disorder,is at risk of developing CF or a CF related disorder, or is a carrier ofa CFTR mutation. In some embodiments, the one or more mutations areselected from Table 1, 2 or 3. In some embodiments, the one or moremutations are selected from Table 1 or 2. In some embodiments, the oneor more mutations are selected from Table 1. In some embodiments, theone or more mutations selected from Table 1, 2, 3, or 4 are part of apanel of CFTR mutations. Also, the system may comprise a device foranalysis and/or interpretation of the data. For example, a computerhaving software to analyze the data for the presence of one of themutations of the invention may be included in the system.

The following embodiments may be used in either the methods or thesystems of the invention. In some embodiments, the sample contains anisolated nucleic acid. In some embodiments, the testing step comprisesnucleic acid sequencing. In some embodiments, the testing step compriseshybridization. In some embodiments, the hybridization is performed usingone or more oligonucleotide probes specific for a region in the CFTRgene (SEQ ID NO:1) (FIG. 1) corresponding to the one or more mutationsselected from Table 1, 2, 3 or 4, and under conditions sufficientlystringent to disallow a single nucleotide mismatch. In some embodiments,the hybridization is performed with a microarray. In some embodiments,the testing step comprises restriction enzyme digestion. In someembodiments, the testing step comprises PCR amplification. In someembodiments, the PCR amplification is digital PCR amplification. In someembodiments, the testing step comprises primer extension. In someembodiments, the primer extension is single-base primer extension. Insome embodiments, the testing step comprises performing a multiplexallele-specific primer extension (ASPE). In yet other embodiments, thetesting step may comprise performing real-time PCR.

In some embodiments, the sample contains purified or partially purifiedprotein. In some embodiments, the testing step comprises amino acidsequencing. For example, in certain embodiments, the system comprises adevice for amino acid sequencing. In some embodiments, the testing stepcomprises performing an immuno assay using one or more antibodies thatspecifically recognize one or more epitopes corresponding to the one ormore mutations selected from Table 1, 2, 3 or 4. In some embodiments,the testing step comprises protease digestion (e.g., trypsin digestion).In some embodiments, the testing step further comprises performing2D-gel electrophoresis.

In some embodiments, the testing step comprises determining the presenceof the one or more mutations using mass spectrometry. In someembodiments, the mass spectrometric format is selected from amongMatrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF),Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), FourierTransform, and combinations thereof.

In some embodiments, the sample is obtained from cells, tissue, wholeblood, mouthwash, plasma, serum, urine, stool, saliva, cord blood,chorionic villus sample, chorionic villus sample culture, amnioticfluid, amniotic fluid culture, transcervical lavage fluid, andcombination thereof. In further embodiments, the sample is obtained froma pregnant woman, for testing the sample for the presence of one or moreCFTR mutations in fetal nucleic acids contained therein. For example, incertain embodiments, the system comprises a station for processing ofthe samples.

In yet another aspect, the present invention provides a method forscreening and/or testing for CFTR mutations, comprising steps of: (a)providing a sample obtained from a subject; (b) testing the sample forthe presence of a mutation at a pre-determined position selected fromTable 1, 2, 3 or 4, in the CFTR gene or protein; and wherein thepresence of the mutation at the pre-determined position indicates thatthe subject has an increased risk of having CF or a CF related disorder,or being a carrier of a CFTR mutation.

Yet other embodiments of the present invention comprise systems forperforming the method. For example, the system may comprise a station ordevice for testing a sample obtained from a subject to determine thepresence of one or more mutations selected from Table 1, 2, 3, or 4 inthe CFTR gene or protein, wherein the presence of the one or moremutations indicates that the subject has CF or a CFTR related disorder,is at risk of developing CF or a CF related disorder, or is a carrier ofa CFTR mutation. In some embodiments, the one or more mutations areselected from Table 1, 2 or 3. In some embodiments, the one or moremutations are selected from Table 1 or 2. In some embodiments, the oneor more mutations are selected from Table 1. In some embodiments, theone or more mutations selected from Table 1, 2, 3, or 4 are part of apanel of CFTR mutations. Also, the system may comprise a device foranalysis and/or interpretation of the data. For example, a computerhaving software to analyze the data for the presence of one of themutations of the invention may be included in the system.

The following embodiments may be used in either the methods or thesystems of the invention. In some embodiments, the testing stepcomprises determining the identity of the nucleotide and/or amino acidat the pre-determined position selected from Table 1, 2, 3 or 4.

In some embodiments, the presence of the mutation is determined bycomparing the identity of the nucleotide and/or amino acid at thepre-determined position to a control.

In some embodiments, the method further comprises a step of determiningif the mutation is listed in Table 1, 2, 3 or 4.

In another aspect, the present invention provides products, e.g.,reagents, for detecting novel CFTR mutations described herein. Suchreagents may be used for detection of the mutations described herein inthe protein sequence and/or the nucleic acid sequence.

In some embodiments, the invention provides a nucleic acid probe thatspecifically binds to a normal CFTR gene but not to a mutant CFTR genecontaining one or more mutations selected from Table 1, 2, 3, or 4. Insome embodiments, the present invention provides a plurality of probes(e.g., as may be used for real-time PCR or sequencing), or an arraycontaining one or more probes that specifically bind to a normal CFTRgene but not to a mutant CFTR gene containing one or more mutationsselected from Table 1, 2, 3, or 4. In some embodiments, the presentinvention provides a nucleic acid probe that specifically binds to amutant CFTR gene containing one or more mutations selected from Table 1,2, 3, or 4 but not to a normal CFTR gene. In some embodiments, the arraycomprises one or more probes that specifically bind to a mutant CFTRgene containing one or more mutations selected from Table 1, 2, 3, or 4but not to a normal CFTR gene.

In some embodiments, the present invention provides an antibody thatspecifically binds to a normal CFTR protein but not to a mutant CFTRprotein containing one or more mutations selected from Table 1, 2, 3, or4. In some embodiments, the present invention provides an antibody thatspecifically binds to a mutant CFTR protein containing one or moremutations selected from Table 1, 2, 3, or 4 but not to a normal CFTRprotein.

In some embodiments, the present invention provides a kit for comprisingone or more reagents that differentiate a normal CFTR gene or proteinfrom a mutant CFTR gene or protein containing one or more mutationsselected from Table 1, 2, 3, or 4. Such kits may be useful, e.g., forscreening and/or testing for CFTR mutations. In some embodiments, theone or more reagents comprises one or more nucleic acid probes. In someembodiments, the one or more reagents comprises one or more antibodies.In some embodiments, the one or more reagents are provided in a form ofmicroarray. In some embodiments, the kit further comprises reagents forprimer extension. Or, probes for the detection of mutations may beprovided. In some embodiments, the kit further comprises a controlindicative of a healthy individual. In some embodiments, the kit furthercomprises an instruction on how to determine if an individual has CF ora CF related disorder, is at risk of developing CF or a CF relateddisorder, or is a carrier of a CFTR mutation.

In still another aspect, the present invention provides a computerreadable medium encoding information corresponding to one or moremutations shown in Tables 1, 2, 3 and 4. Such computer readable mediamay be part of the systems as described herein.

Other features, objects, and advantages of the present invention areapparent in the detailed description and claims that follow. It shouldbe understood, however, that the detailed description, the drawings, andthe claims, while indicating embodiments of the present invention, aregiven by way of illustration only, not limitation. Various changes andmodifications within the scope of the invention will become apparent tothose skilled in the art.

FIGURES

FIG. 1 is a genomic sequence of the CFTR gene according to an embodimentof the invention.

FIG. 2 is a cDNA sequence of CFTR according to an embodiment of theinvention.

FIG. 3 is an amino acid sequence of CFTR according to an embodiment ofthe invention.

FIG. 4 is a nucleotide sequence of the 5′ end of the CFTR gene accordingto an embodiment of the invention.

FIG. 5 is is a schematic of a system according to an embodiment of theinvention.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

Antibody: As used herein, the term “antibody” refers to a polypeptideconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes or fragments of immunoglobulin genes. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are typicallyclassified as either kappa or lambda. Heavy chains are typicallyclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Atypical immunoglobulin (antibody) structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(VL) and “variable heavy chain” (VH) refer to these light and heavychains respectively. An antibody can be specific for a particularantigen. The antibody or its antigen can be either an analyte or abinding partner. Antibodies exist as intact immunoglobulins or as anumber of well-characterized fragments produced by digestion withvarious peptidases. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′2, a dimerof Fab which itself is a light chain joined to VH-CH1 by a disulfidebond. The F(ab)′2 may be reduced under mild conditions to break thedisulfide linkage in the hinge region thereby converting the (Fab′)2dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see, Fundamental Immunology, W. E. Paul, ed.,Raven Press, N.Y. (1993), for a more detailed description of otherantibody fragments). While various antibody fragments are defined interms of the digestion of an intact antibody, one of ordinary skill inthe art will appreciate that such Fab′ fragments may be synthesized denovo either chemically or by utilizing recombinant DNA methodology.Thus, the term “antibody,” as used herein also includes antibodyfragments either produced by the modification of whole antibodies orsynthesized de novo using recombinant DNA methodologies. In someembodiments, antibodies are single chain antibodies, such as singlechain Fv (scFv) antibodies in which a variable heavy and a variablelight chain are joined together (directly or through a peptide linker)to form a continuous polypeptide. A single chain Fv (“scFv”) polypeptideis a covalently linked VH::VL heterodimer which may be expressed from anucleic acid including VH- and VL-encoding sequences either joineddirectly or joined by a peptide-encoding linker. (See, e.g., Huston, etal. (1988) Proc. Nat. Acad. Sci. USA, 85:5879-5883, the entire contentsof which are herein incorporated by reference.) A number of structuresexist for converting the naturally aggregated, but chemically separatedlight and heavy polypeptide chains from an antibody V region into anscFv molecule which will fold into a three dimensional structuresubstantially similar to the structure of an antigen-binding site. See,e.g. U.S. Pat. Nos. 5,091,513 and 5,132,405 and 4,956,778.

Allele: As used herein, the term “allele” refers to different versionsof a nucleotide sequence of a same genetic locus (e.g., a gene).

Allele specific primer extension (ASPE): As used herein, the term“allele specific primer extension (ASPE)” refers to a mutation detectionmethod utilizing primers which hybridize to a corresponding DNA sequenceand which are extended depending on the successful hybridization of the3′ terminal nucleotide of such primer. Typically, extension primers thatpossess a 3′ terminal nucleotide which form a perfect match with thetarget sequence are extended to form extension products. Modifiednucleotides can be incorporated into the extension product, suchnucleotides effectively labeling the extension products for detectionpurposes. Alternatively, an extension primer may instead comprise a 3′terminal nucleotide which forms a mismatch with the target sequence. Inthis instance, primer extension does not occur unless the polymeraseused for extension inadvertently possesses exonuclease activity.

Amplification: As used herein, the term “amplification” refers to anymethods known in the art for copying a target nucleic acid, therebyincreasing the number of copies of a selected nucleic acid sequence.Amplification may be exponential or linear. A target nucleic acid may beeither DNA or RNA. Typically, the sequences amplified in this mannerform an “amplicon.” Amplification may be accomplished with variousmethods including, but not limited to, the polymerase chain reaction(“PCR”), transcription-based amplification, isothermal amplification,rolling circle amplification, etc. Amplification may be performed withrelatively similar amount of each primer of a primer pair to generate adouble stranded amplicon. However, asymmetric PCR may be used to amplifypredominantly or exclusively a single stranded product as is well knownin the art (e.g., Poddar et al. Molec. And Cell. Probes 14:25-32(2000)). This can be achieved using each pair of primers by reducing theconcentration of one primer significantly relative to the other primerof the pair (e.g., 100 fold difference). Amplification by asymmetric PCRis generally linear. Additionally, methods such as real-time PCR may beutilized. A skilled artisan will understand that different amplificationmethods may be used together.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biological sample: As used herein, the term “biological sample”encompasses any sample obtained from a biological source. A biologicalsample can, by way of non-limiting example, include blood, amnioticfluid, sera, urine, feces, epidermal sample, skin sample, cheek swab,sperm, amniotic fluid, cultured cells, bone marrow sample and/orchorionic Convenient biological samples may be obtained by, for example,scraping cells from the surface of the buccal cavity. The termbiological sample encompasses samples which have been processed torelease or otherwise make available a nucleic acid or protein fordetection as described herein. For example, a biological sample mayinclude a cDNA that has been obtained by reverse transcription of RNAfrom cells in a biological sample. The biological sample may be obtainedfrom a stage of life such as a fetus, young adult, adult, and the like.Fixed or frozen tissues also may be used.

Carrier: The term “carrier,” as used in the context of CF, refers to aperson who is symptom-free but carries a CFTR mutation that can bepassed to his/her children. Typically, a carrier has one CFTR allelethat contains a disease causing mutation and a second allele that isnormal or not disease-related. CF and CF related disorders are“autosomal recessive” diseases, meaning that a mutation produces littleor no phenotypic effect when present in a heterozygous configurationwith a non-disease related allele, but produces a “disease state” when aperson is homozygous, i.e., both CFTR alleles are mutant alleles thatcontain the same disease causing mutation or compound heterozygous,i.e., both CFTR alleles are mutant alleles that contain two differentdisease-causing mutations. A carrier status is whether or not one is acarrier.

Coding sequence vs. non-coding sequence: As used herein, the term“coding sequence” refers to a sequence of a nucleic acid or itscomplement, or a part thereof, that can be transcribed and/or translatedto produce the mRNA for and/or the polypeptide or a fragment thereof.Coding sequences include exons in a genomic DNA or immature primary RNAtranscripts, which are joined together by the cell's biochemicalmachinery to provide a mature mRNA. The anti-sense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom. As used herein, the term “non-coding sequence” refersto a sequence of a nucleic acid or its complement, or a part thereof,that is not transcribed into amino acid in vivo, or where tRNA does notinteract to place or attempt to place an amino acid. Non-codingsequences include both intron sequences in genomic DNA or immatureprimary RNA transcripts, and gene-associated sequences such aspromoters, enhancers, silencers, etc.

Complement: As used herein, the terms “complement,” “complementary” and“complementarity,” refer to the pairing of nucleotide sequencesaccording to Watson/Crick pairing rules. For example, a sequence5′-GCGGTCCCA-3′ has the complementary sequence of 5′-TGGGACCGC-3′. Acomplement sequence can also be a sequence of RNA complementary to theDNA sequence. Certain bases not commonly found in natural nucleic acidsmay be included in the complementary nucleic acids including, but notlimited to, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), andPeptide Nucleic Acids (PNA). Complementary need not be perfect; stableduplexes may contain mismatched base pairs, degenerative, or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength andincidence of mismatched base pairs.

Control: As used herein, the term “control” has its art-understoodmeaning of being a standard against which results are compared.Typically, controls are used to augment integrity in experiments byisolating variables in order to make a conclusion about such variables.In some embodiments, a control is a reaction or assay that is performedsimultaneously with a test reaction or assay to provide a comparator. Inone experiment, the “test” (i.e., the variable being tested) is applied.In the second experiment, the “control,” the variable being tested isnot applied. In some embodiments, a control is a historical control(i.e., of a test or assay performed previously, or an amount or resultthat is previously known). In some embodiments, a control is orcomprises a printed or otherwise saved record. A control may be apositive control or a negative control.

Crude: As used herein, the term “crude,” when used in connection with abiological sample, refers to a sample which is in a substantiallyunrefined state. For example, a crude sample can be cell lysates orbiopsy tissue sample. A crude sample may exist in solution or as a drypreparation.

Deletion: As used herein, the term “deletion” encompasses a mutationthat removes one or more nucleotides from a naturally-occurring nucleicacid.

Epitope: As used herein, the term “epitope” refers to a fragment orportion of a molecule or a molecule compound (e.g., a polypeptide or aprotein complex) that makes contact with a particular antibody orantibody like proteins.

Familial history: As used herein, the term “familial history” typicallyrefers to occurrence of events (e.g., CF disease, CF related disorder orCFTR mutation carrier) relating to an individual's immediate familymembers including parents and siblings. Sometimes, family history alsomay include grandparents.

Flanking: As used herein, the term “flanking” is meant that a primerhybridizes to a target nucleic acid adjoining a region of interestsought to be amplified on the target. The skilled artisan willunderstand that preferred primers are pairs of primers that hybridize 3′from a region of interest, one on each strand of a target doublestranded DNA molecule, such that nucleotides may be add to the 3′ end ofthe primer by a suitable DNA polymerase. For example, primers that flankmutant CTFR sequences do not actually anneal to the mutant sequence butrather anneal to sequence that adjoins the mutant sequence. In somecases, primers that flank a CFTR exon are generally designed not toanneal to the exon sequence but rather to anneal to sequence thatadjoins the exon (e.g. intron sequence). However, in some cases,amplification primer may be designed to anneal to the exon sequence.

Genotype: As used herein, the term “genotype” refers to the geneticconstitution of an organism. More specifically, the term refers to theidentity of alleles present in an individual. “Genotyping” of anindividual or a DNA sample refers to identifying the nature, in terms ofnucleotide base, of the two alleles possessed by an individual at aknown polymorphic site.

Heterozygous: As used herein, the term “heterozygous” or “HET” refers toan individual possessing two different alleles of the same gene. As usedherein, the term “heterozygous” encompasses “compound heterozygous” or“compound heterozygous mutant.” As used herein, the term “compoundheterozygous” refers to an individual possessing two different alleles.As used herein, the term “compound heterozygous mutant” refers to anindividual possessing two different copies of an allele, such allelesare characterized as mutant forms of a gene. The term “mutant” as usedherein refers to a mutated, or potentially non-functional form of agene. (See “mutations of the CFTR gene.”)

Homozygous: As used herein, the term “homozygous” refers to anindividual possessing two copies of the same allele. As used herein, theterm “homozygous mutant” refers to an individual possessing two copiesof the same allele, such allele being characterized as the mutant formof a gene. The term “mutant” as used herein refers to a mutated, orpotentially non-functional form of a gene.

Hybridize: As used herein, the term “hybridize” or “hybridization”refers to a process where two complementary nucleic acid strands annealto each other under appropriately stringent conditions. Oligonucleotidesor probes suitable for hybridizations typically contain 10-100nucleotides in length (e.g., 18- 50, 12-70, 10-30, 10-24, 18-36nucleotides in length). Nucleic acid hybridization techniques are wellknown in the art. See, e.g., Sambrook, et al., 1989, Molecular Cloning:A Laboratory Manual, Second Edition, Cold Spring Harbor Press,Plainview, N.Y. Those skilled in the art understand how to estimate andadjust the stringency of hybridization conditions such that sequenceshaving at least a desired level of complementary will stably hybridize,while those having lower complementary will not. For examples ofhybridization conditions and parameters, see, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994,Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus,N.J.

Insertion or addition: As used herein, the term “insertion” or“addition” refers to a change in an amino acid or nucleotide sequenceresulting in the addition of one or more amino acid residues ornucleotides, respectively, as compared to the naturally occurringmolecule.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism such as a non-human animal.

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, substantially 100%, or 100% pure. Asused herein, a substance is “pure” if it is substantially free of othercomponents. As used herein, the term “isolated cell” refers to a cellnot contained in a multi-cellular organism.

Labeled: The terms “labeled” and “labeled with a detectable agent ormoiety” are used herein interchangeably to specify that an entity (e.g.,a nucleic acid probe, antibody, etc.) can be visualized, for examplefollowing binding to another entity (e.g., a nucleic acid, polypeptide,etc.). The detectable agent or moiety may be selected such that itgenerates a signal which can be measured and whose intensity is relatedto (e.g., proportional to) the amount of bound entity. A wide variety ofsystems for labeling and/or detecting proteins and peptides are known inthe art. Labeled proteins and peptides can be prepared by incorporationof, or conjugation to, a label that is detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical,chemical or other means. A label or labeling moiety may be directlydetectable (i.e., it does not require any further reaction ormanipulation to be detectable, e.g., a fluorophore is directlydetectable) or it may be indirectly detectable (i.e., it is madedetectable through reaction or binding with another entity that isdetectable, e.g., a hapten is detectable by immunostaining afterreaction with an appropriate antibody comprising a reporter such as afluorophore). Suitable detectable agents include, but are not limitedto, radionucleotides, fluorophores, chemiluminescent agents,microparticles, enzymes, colorimetric labels, magnetic labels, haptens,molecular beacons, aptamer beacons, and the like.

Multiplex PCR: As used herein, the term “multiplex PCR” refers toamplification of two or more regions which are each primed using adistinct primers pair.

Multiplex ASPE: As used herein, the term “multiplex ASPE” refers to anassay combining multiplex PCR and allele specific primer extension fordetecting polymorphisms. Typically, multiplex PCR is used to firstamplify regions of DNA that will serve as target sequences for ASPEprimers. See the definition of allele specific primer extension.

Mutations of the CFTR gene: As used herein, the term “mutations of theCFTR gene” refers to one or more abnormal nucleic acid sequences ascompared to a wild-type CFTR gene sequence. The “mutations of the CFTRgene” are also referred to as “mutant CF sequences.” Mutations of theCFTR gene encompass substitutions (e.g., single nucleotide polymorphisms(SNP)), deletions, insertions, additions, and/or duplications.

Primer: As used herein, the term “primer” refers to a shortsingle-stranded oligonucleotide capable of hybridizing to acomplementary sequence in a nucleic acid sample. Typically, a primerserves as an initiation point for template dependent DNA synthesis.Deoxyribonucleotides can be added to a primer by a DNA polymerase. Insome embodiments, such deoxyribonucleotides addition to a primer is alsoknown as primer extension. The term primer, as used herein, includes allforms of primers that may be synthesized including peptide nucleic acidprimers, locked nucleic acid primers, phosphorothioate modified primers,labeled primers, and the like. A “primer pair” or “primer set” for a PCRreaction typically refers to a set of primers typically including a“forward primer” and a “reverse primer.” As used herein, a “forwardprimer” refers to a primer that anneals to the anti-sense strand ofdsDNA. A “reverse primer” anneals to the sense-strand of dsDNA.

Polymorphism: As used herein, the term “polymorphism” refers to thecoexistence of more than one form of a gene or portion thereof.

Pure or substantially pure: As used herein, the term “pure orsubstantially pure” refers to a compound, e.g., a protein or polypeptidethat has been separated from components which naturally accompany it.Typically, a compound is substantially pure when at least 10%, morepreferably at least 20%, more preferably at least 50%, more preferablyat least 60%, more preferably at least 75%, more preferably at least90%, and most preferably at least 99% of the total material (by volume,by wet or dry weight, or by mole percent or mole fraction) in a sampleis the compound of interest. Purity can be measured by any appropriatemethod, e.g., in the case of polypeptides by column chromatography, gelelectrophoresis or HPLC analysis. A compound, e.g., a protein, is alsosubstantially purified when it is essentially free of naturallyassociated components or when it is separated from the nativecontaminants which accompany it in its natural state.

Real-time PCR: As used herein, the term “real-time PCR” refers toquantitative real time polymerase chain reaction (Q-PCR/qPCR/qrt-PCR) orkinetic polymerase chain reaction (KPCR), is a laboratory techniquebased on the PCR, which is used to amplify and simultaneously quantify atargeted DNA molecule. It enables both detection and quantification (asabsolute number of copies or relative amount when normalized to DNAinput or additional normalizing genes) of one or more specific sequencesin a DNA sample.

Sense strand vs. anti-sense strand: As used herein, the term “sensestrand” refers to the strand of double-stranded DNA (dsDNA) thatincludes at least a portion of a coding sequence of a functionalprotein. As used herein, the term “anti-sense strand” refers to thestrand of dsDNA that is the reverse complement of the sense strand.

Specific: As used herein, the term “specific,” when used in connectionwith an oligonucleotide primer, refers to an oligonucleotide or primer,under appropriate hybridization or washing conditions, is capable ofhybridizing to the target of interest and not substantially hybridizingto nucleic acids which are not of interest. Higher levels of sequenceidentity are preferred and include at least 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% sequence identity. In some embodiments,a specific oligonucleotide or primer contains at least 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, ormore bases of sequence identity with a portion of the nucleic acid to behybridized or amplified when the oligonucleotide and the nucleic acidare aligned.

Subject: As used herein, the term “subject” refers to a human or anynon-human animal. A subject can be a patient, which refers to a humanpresenting to a medical provider for diagnosis or treatment of adisease. A human includes pre and post natal forms. Particularlypreferred subjects are humans being tested for the existence of a CFTRcarrier state, CF disease or CF related disorder state.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially complementary: As used herein, the term “substantiallycomplementary” refers to two sequences that can hybridize understringent hybridization conditions. The skilled artisan will understandthat substantially complementary sequences need not hybridize alongtheir entire length. In some embodiments, “stringent hybridizationconditions” refer to hybridization conditions at least as stringent asthe following: hybridization in 50% formamide, 5×SSC, 50 mM NaH₂PO₄, pH6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5×Denhart'ssolution at 42° C., overnight; washing with 2×SSC, 0.1% SDS at 45° C.;and washing with 0.2×SSC, 0.1% SDS at 45° C. In some embodiments,stringent hybridization conditions should not allow for hybridization oftwo nucleic acids which differ over a stretch of 20 contiguousnucleotides by more than two bases.

Substitution: As used herein, the term “substitution” refers to thereplacement of one or more amino acids or nucleotides by different aminoacids or nucleotides, respectively, as compared to the naturallyoccurring molecule.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of the disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition may not exhibitsymptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition will develop the disease, disorder, and/or condition.In some embodiments, an individual who is susceptible to a disease,disorder, and/or condition will not develop the disease, disorder,and/or condition.

Wild-type: As used herein, the term “wild-type” refers to the typical orthe most common form existed in nature. For example, a wild-type CFTRgene or protein refers to the typical or the most common form of CFTRgene or protein existed in a natural population. As used herein,“wild-type” is used interchangeably with “naturally-occurring.” In someembodiment, a wild-type CFTR gene or a locus thereof, refers to the CFTRgene sequence which is found in NCBI GenBank locus ID M58478 (HUMCFTC)(SEQ ID NO:4) (FIG. 4). The CFTR gene is located on chromosome 7, whichmay be found in NCBI GenBank locus AC000111 and AC000061, the contentsof which are incorporated herein in their entirety by reference. ThecDNA for the CFTR gene is found in Audrezet et al., Hum. Mutat. (2004)23 (4), 343-357.

DETAILED DESCRIPTION

The present invention provides, among other things, methods, productsand systems that use novel mutations in the cystic fibrosistransmembrane conductance regulator (CFTR) gene in screening and/ortesting for CF and CF related diseases, disorders or conditions. Forexample, the novel mutations provided herein can be used to assist inclinical diagnosis of CF disease, CF related disease, disorder orcondition, or carrier status and for genetic counseling (e.g., forevaluation of an individual's risk for developing CF or being a carrierof a CFTR mutation). The novel mutations provided herein can be usedalone or in combination with other known CFTR mutations as part of apanel of CFTR mutations.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Novel Mutations in the CFTR Gene

The CFTR gene was mapped to chromosome 7 and described in, for example,U.S. Pat. No. 6,201,107 and U.S. Pat. No. 5,776,677, the disclosures ofwhich are incorporated by reference herein in their entirety. The CFTRgenomic sequence is described in GenBank Accession Number NC_000007(range: 117120016 . . . 117308718; the entire contents of which areherein incorporated by reference) (SEQ ID NO:1) (FIG. 1). The CFTR genecontains 27 exons. The exons are numbered 1, 2, 3, 4, 5, 6a, 6b, 7, 8,9, 10, 11, 12, 13, 14a, 14b, 15, 16, 17a, 17b, 18, 19, 20, 21, 22, 23,and 24. The CFTR cDNA sequence is described in GenBank Accession NumberAR016032.1 (SEQ ID NO:2) (FIG. 2).

The CFTR protein is described in, for example, U.S. Pat. No. 5,543,399,the disclosure of which is incorporated by reference herein in itsentirety. The CFTR protein sequence is also described in GenBankAccession Number AAC90840.1 (SEQ ID NO:3) (FIG. 3).

As described in Example 1, the inventors of the present applicationidentified various novel mutations in the CFTR gene (Table 5). Thesemutations were identified by sequence analysis of the CFTR gene inspecimens submitted for clinical testing obtained from individuals whowere known to be affected with CF or likely to be a carrier because offamilial history, or suspected to be affected with CF based on other CFtesting (see Clinical Indication listed in Table 5). The mutations wereidentified by comparing the CFTR gene sequence from patient samples tothe wild-type CFTR gene or protein sequence (see SEQ ID NO:1-3). Asshown in Table 5, patients carrying these mutations were from differentethnic groups including Caucasians, African Americans, Hispanics, andAsians. Thus, these mutations may be particularly useful for developingmore effective genetic testing for patients from non-Caucasian racialgroups.

Novel mutations described herein are located in introns (e.g., intron 3,intron 6a, intron 11, intron 14a, intron 19, intron 20, intron 21, andintron 23) and exons (e.g., exon 2, exon 3, exon 4, exon 5, exon 6a,exon 6b, exon 7, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14a,exon 14b, exon 15, exon 16, exon 17a, exon 17b, exon 19, exon 20, exon21, exon 22, and exon 24). Some of the novel mutations are nonsensemutations, i.e., mutations that result in a stop codon. Some of thenovel mutations are missense mutations, i.e., mutations that result inamino acid substitutions. Some of the novel mutations cause in-frameinsertions and/or deletions. Some of the novel mutations delete one ormore nucleotides in such a manner as to lead to a shift in the readingframe. Some of the novel mutations alter the sequence at a splicejunction, for example, consensus splice site ag/gt or other splicesites. Thus, most of the novel mutations described herein are likely todisrupt CFTR gene or protein expression or function.

The “ACMG recommendations for standards for interpretation and reportingof sequence variations: Revisions 2007” (Richards S C et al. Genetics inMedicine, 10:294-300, which is incorporated herein by reference),provides interpretive categories and definitions of sequence variationswhich can be used, along with additional test results and clinicalinformation to classify the novel mutations described herein into thefollowing groups.

Group I: Patient has a novel sequence change that can be classified ascategory 2 according to the ACMG guidelines (i.e., nonsense, frame shift(FS), consensus splice site ag/gt). Patient has another well establishedCF disease causing mutation (i.e. F508, W1282X, etc). Patient indicationis suspected of having CF, known to be affected with CF or identifiedthrough newborn screening. The Group I mutations, which are ofparticular interest, are shown in Table 1 and these mutations areexpected to cause CF or CF related diseases, disorders or conditions.

Group IIA: Patient has novel sequence change that can be classified ascategory 3 according to the ACMG guidelines (i.e., missense, in-frameins/del, other splice site mutations, etc). Patient has another wellestablished CF disease causing mutation (i.e., F508, W1282X, etc.)Patient is suspected of having CF, known to be affected with CF, oridentified through positive newborn screening. The Group IIA mutationsare shown in Table 2 (under subsection Group IIA).

Group IIB: Patent has a novel sequence change that can be classified ascategory 3 according to the ACMG guidelines (i.e., missense, in-frameins/del, other splice site mutations, etc). Patient is suspected ofhaving CF, known to be affected with CF, or identified through positivenewborn screening. The Group JIB mutations are shown in Table 2 (undersubsection Group IIB).

Group III: Patient has novel sequence change that can be classified ascategory 3 according to the ACMG guidelines (i.e., missense, in-frameinsertions/deletions, other splice site mutations, etc). Patient hasanother well established CF disease causing mutation (i.e. F508, W1282X,etc). Patient indication is suspected of having CF, known to be affectedwith CF, or identified through newborn screening. Patient has anadditional change(s) of unknown clinical significance. The Group IIImutations are shown in Table 3.

Group IV: Mutations other than the Group I, II, and III mutationsidentified above. The Group IV mutations are shown in Table 4.

Novel CFTR mutations according to the invention however are not limitedto the specific nucleotide or amino acid variations identified in Tables1-4 and should encompass any abnormal nucleotides or amino acidresidues, as compared to the wild-type CFTR gene or protein sequences,that may be present at any of the positions identified in Tables 1-4.

TABLE 1 Group I mutations Sequence Type of AA Other Change mutationChange Ethnicity Mutations Clinical Information Exon 1824delA Frame- n/aCaucasian F508del Mutation was identified in a 22 year old e12 shiftpatient with a known diagnosis of CF. (FS)This patient carried a second mutation known to cause CF (F508del).2957delT FS n/a Caucasian F508delMutation was identified in a 1 year old e15patient with a known diagnosis of CF.The patient carried a second mutation known to cause CF (F508del).4089ins4 FS n/a Caucasian F508delMutation was identified in a 7 year old e21patient with a known diagnosis of CF.The patient had a positive sweat chloridetest. The patient carried a second mutation known to cause CF (F508del).4374 + Splice n/a 1.  1.  Patient #1: Mutation was identified in a i232T > C site Caucasian F508del 45 year old patient with a suspectedmutation 2. 2.  diagnosis of CF. The patient carried a Caucasian F508delsecond mutation known to cause CF (F508del).Patient #2: Mutation was identified in a52 year old patient with a suspecteddiagnosis of CF. The patient carried a second mutation known to cause CF(F508del). 3064A > T Nonsense K978X African Q1042XMutation was identified in a 26 year old e16 Americanpatient with a known diagnosis of CF.The patient carried a second mutation likely to cause CF Q1042X. 246C >G Nonsense Y38X Caucasian F508delMutation was identified in a 1 month old e2patient with a suspected diagnosis of CF.The patient had a positive sweat chloridetest. The patient carried a second mutation known to cause CF (F508del).

TABLE 2 Group II Mutations Sequence Type of AA Other Clinical Changemutation Change Ethnicity Mutations Information Exon Group II A 269C > TMissense (MS) A46V 1) Caucasian 1) Patient #1: Mutation was e2 3849 +1219 identified in a 32 year old 2G > A patient who was tested due to 2)Black 2) F508del abnormalities found on fetal 3) African 3) noneultrasound. The patient American carried a second mutation of unknownclinical significance (3849 + 12192G > A). Patient #2: Mutation wasidentified in a 2 month old patient who was tested based on follow-upfor a positive newborn screen. The patient carried a second mutationknown to cause cystic fibrosis (F508del). Patient #3: Mutation wasidentified in a 24 year old patient who was tested as a parentalfollow-up to a positive newborn screen. 2902 G > T MS D924Y CaucasianF508del Mutation was identified in a e15 1 month old patient with asuspected diagnosis of CF. The patient also had a positive sweatchloride test and carried a second mutation known to cause CF (F508del)3814G > A MS E1228K Caucasian F508del Mutation was identified in a e19 1month old patient with a suspected diagnosis of CF. The patient had aborderline sweat chloride test. The patient carried a second mutationknown to cause CF (F508del). 502G > C MS G124R Not Provided F508delMutation was identified in a e4 2 month old patient with a suspecteddiagnosis of CF. The patient had a positive sweat chloride test. Thepatient carried a second mutation known to cause CF (F508del). 1520G > TMS G463V Caucasian F508del Mutation was identified in a e9 17 year oldpatient with a known diagnosis of CF. Patient carried a second mutationknown to cause CF (F508del). 511_513dup In frame L127dup Caucasian,W1282X Mutation was identified in a e4 TTA duplication Asian newbornwith a suspected diagnosis of CF. The patient had clinical symptoms ofCF including as a positive sweat chloride test, meconium ileus,echogenic bowel, and pancreatic insufficiency. The patient carried asecond mutation known to cause CF (W1282X). 978A > T MS E282D 1. notprovided 1. Patient #1: Mutation was e6b 3120 + 1G > identified in a 10year old A patient with a suspected 2. not provided 2) none diagnosis ofCF. The patient had a positive sweat chloride test. The patient carrieda second mutation known to cause CF (3120 + 1G > A). Patient #2:Mutation was identified in a 4 year old patient with a suspecteddiagnosis of CF and a family history of CF. 843G > C MS Q237H CaucasianF508del Mutation was identified in a e6a 2 month old patient with aknown diagnosis of CF. The patient carried a second mutation known tocause CF (F508del). 829C > T MS L233F Caucasian D1152H Mutation wasidentified in a e6a 1 month old patient who was tested following apositive newborn screen. The patient carried a second mutation known tocause CF (D1152H). 4096-6C > T Splice site None Caucasian F508delMutation was identified in a i21 mutation 58 year old patient with asuspected diagnosis of CF. The patient carried a second mutation knownto cause CF (F508del). 4375-7delT Splice site None Caucasian F508delMutation was identified in a i23 mutation 6 year old patient with asuspected diagnosis of CF. Patient has a family history, a borderlinesweat chloride test and recurrent pneumonia. The patient carried asecond mutation known to cause CF (F508del). 1586 G > C MS 5485TCaucasian S1235R Mutation was identified in a e10 2 year old patientwith a suspected diagnosis of CF. The patient carried a second mutation51235R (3837T > G) which has been reported in individuals with varyingCF phenotypes. Group II B 875 + 4G > T Splice site n/a African noneMutation was i6a mutation American identified in a 1 month old patientwho had a positive newborn screening test. 4005 + 3G > T Splice site n/aCaucasian none Mutation was identified in a i20 mutation 40 year oldpatient who was tested to determine if they were a carrier, there was nofamily history of CF.

TABLE 3 Group III Mutations Sequence Type of AA Other Change mutationChange Ethnicity Mutations Clinical Information Exon 2711T > C MS 1860TCaucasian F508del, Mutation was identified in a 58 year old e14a E528Ewoman with a suspected diagnosis of CF.The patient carried a second mutation known to cause CF (F508del) and anadditional mutation of unknown clinical significance (E528E).. 3891G > CMS L1253F Not  G85E, L15P Mutation was identified in a 32 year old e20provided patient with a known diagnosis of CF.The patient carried a second mutation known to cause CF (G85E) and anadditional mutation of unknown clinical significance (L15P). 2524C > TMS P798S African F508del, Mutation was identified in a 5 year old e13American R74W, patient with a suspected diagnosis of CF. G921E,The patient had a positive sweat chloride D1270Ntest. This patient carried a second mutation known to cause CF (F508del)and three additional mutations of unknown clinical significance (R74W,G921E, D1270N). 2894G > A MS G921E African F508del,Mutation was identified in a 5 year old e15 American R74W,patient with a suspected diagnosis of CF. P798S,The patient had a positive sweat chloride D1270Ntest. This patient carried a second mutation known to cause CF (F508del)and three additional mutations of unknown clinical significance (R74W,P789S, D1270N).

TABLE 4 Group IV Mutations Sequence Type of AA Other Change mutationChange Ethnicity Mutations Clinical Information Exon 405 + Possible n/aCaucasian F508del Mutation was identified in a 35 year old i3 10247C  >T splice  patient who was tested to determine if sitethey were a carrier, there was no family mutationhistory of CF. This patient carried a second mutation known to cause CF(F508del). 405 + 10255 Possible n/a Not  F508del,Mutation was identified in a 10 year old i3 delC splice  Provided124del23bp patient. The patient carries two mutations siteknow to cause CF (F508del and mutation 124del23). 1811 + Possible n/a 1.1. F508del Patient #1: Mutation was identified in a 1 i11 1643G > Tsplice  Hispanic year old patient with a known diagnosis site 2. 2. F508del of CF. Patient had a positive sweat mutation Hispanicchloride test. The patient carried a 3. Not 3. nonesecond mutation known to cause CF provided (F508del).Patient #2: Mutation was identified in a 6year old patient with a known diagnosisof CF. The patient carried a second mutation know to cause CF (F508del).Patient #3: Mutation was identified in an8 month old patient with a suspected diagnosis of CF. 1812- Splice n/aCaucasian none Mutation was identified in a 15 year old i11 13A > G sitepatient with a suspected diagnosis of CF. mutationThe patient has chronic sinusitis. 2752- Possible n/a African F693LMutation was identified in a 6 year old i14a 33insA splice Americanpatient with a known diagnosis of CF. siteThe patient carries a second mutation of mutationunknown clinical significance (F693L). 3849 + Possible n/a CaucasianA46V Mutation was identified in a 32 year old i19 12192G > A splicepatient who was tested due to siteabnormalities found on fetal ultrasound. mutationThe patient carried an additionalmutation of known clinical significance (A46V). 724G > A MS A198THispanic none Mutation was identified in a 4 month old e6apatient with a suspected diagnosis of CF. 3899C > T MS A1256V Guyanesenone Mutation was identified in a 45 year old e20patient who was tested to determine ifthey were a carrier, there was no family history of CF. 3986C > T MSA1285V Not  none Mutation was identified in a 23 year old e20 Providedpatient who was tested to determine ifthey were a carrier, there was no family history of CF. 901G> A MS E257KHispanic none Mutation was identified in a 4 year old e6bpatient with a suspected diagnosis of CF.The patient has asthma and recurring pneumonia. 392 T > C MS F875 Not none The mutation was identified in a 1 month e3 Providedold patient with a suspected diagnosis of CF. 3463T > C MS F1111LHispanic none Mutation was identified in a 6 year old e17bpatient with a suspected diagnosis of CF. The patient has asthma 1757G >A MS G542E Hispanic none Mutation was identified in a 25 year old e11patient who was tested to determine ifthey were a carrier, there was no familyhistory of CF. The patient carried 2 copies of G542E.. 4025G > C MSG1298A Asian G970D, Mutation was identified in a 34 year old e21 Q1352Hpatient with congenital absence of thevas deferens. The patient carried twoother mutations of unknown clinical significance (G970D and Q1352H)4129G > T MS G1333W Not  none Mutation was identified in an 8 year olde22 Provided patient with a suspected diagnosis of CF.Patient had recurrent respiratory infections and chronic cough. 663T > GMS I177M Caucasian none Mutation was identified in a 34 year old e5patient who was tested to determine ifthey were a carrier, there was no family history of CF. 3200T > C MSI1023T Hispanic none Mutation was identified in a 34 year old e17apatient who was tested to determine ifthey were a carrier, there was no family history of CF. 4412T > C MSI1427T Asian S1444S Mutation was identified in a 34 year old e24patient who was tested to determine ifthey were a carrier, there was no familyhistory of CF. The patient carried anothermutation that is considered likely to be clinically benign (S144S).620A > C MS K163T Caucasian noneMutation was identified in a 32 year old e4patient with a family history of CF. 1738 A > G MS K536 Hispanic I488IMutatin was identified in a 19  e11 year old patient who's son had a positive newborn screening test. The patient carried anothermutation that is considered likely to be clinically benign (I1488I)..3370A > C MS K1080Q Caucasian none Mutation was identifed in a 9 e17byear old patient with a suspected diagnosis of CF. The patiend hadasthma and failure to thrive. 1129 C > T MS L33F Asian noneMutation was identifed in a 37 e7 year old patient who tested todetermind if they were a carrier, there was no family history of CF.2383C > T MS R751C Caucasian 2183 Mutation was identifed in a 36 e13delAA > G yar old patient who was beingtested due to a partner being a CF carrier. The patiend also carried a second mutation known to cause CF (2183delAA > G). 2761delTCT In frameS877del Caucasian F508del, Mutation was identied in 1 e14b deletionC1152H month old patient who had a  positive sweat chloride test. Thepatient carried two additional mutations known to cause CF(F508del and D1125H). 1106A > G MS Y325C Caucasian R334WMutation was identifed in a 35 c7 year old patient who was tested todetermine if they were a carrier, there was no family history of CF.Patient carried a second mutation known to cause CF (R334W). 622A > G MST164A Caucasian none Mutation was identiied in a 3 e5month old patient with a suspected diagnosis of CF.Detection of CFTR Mutations

A variety of methods known in the art can be used to detect CFTR genemutations disclosed in the present invention. For example, methods thathave been used to detect previously identified CFTR gene mutations havebeen described and are adaptable for use with the present invention. Seee.g., Audrezet et al., “Genomic rearrangements in the CFTR gene:extensive allelic heterogeneity and diverse mutational mechanisms” HumMutat. 2004 April; 23(4):343-57; PCT WO 2004/040013 A1 and correspondingUS application No. 20040110138; titled “Method for the detection ofmultiple genetic targets” by Spiegelman and Lem; US patent applicationNo. 20030235834; titled “Approaches to identify cystic fibrosis” byDunlop et al.; and US patent application No. 20040126760 titled “Novelcompositions and methods for carrying out multiple PCR reactions on asingle sample” by N. Broude, the entire contents of each of which areherein incorporated by reference.

Nucleic Acid Analyses

In certain embodiments, CFTR gene mutations disclosed herein aredetected at the nucleic acid level. For example, nucleic acid can beanalyzed by sequencing, hybridization, PCR amplification, restrictionenzyme digestion, primer extension such as single-base primer extensionor multiplex allele-specific primer extension (ASPE).

Nucleic acid analyses can be performed on genomic DNA, messenger RNAs,and/or cDNA. In many embodiments, nucleic acids are extracted from abiological sample. In some embodiments, nucleic acids are analyzedwithout having been amplified. In some embodiments, nucleic acids areamplified using techniques known in the art (such as polymerase chainreaction (PCR)) and amplified nucleic acids are used in subsequentanalyses. Multiplex PCR, in which several amplicons (e.g., fromdifferent genomic regions) are amplified at once using multiple sets ofprimer pairs, may be employed. Additionally, methods such as real-timePCR, as are known in the art, may be used to perform nucleic acidanalysis.

In some embodiments, nucleic acids are amplified in a manner such thatthe amplification product for a wild-type allele differs in size fromthat of a mutant allele. Thus, presence or absence of a particularmutant allele can be determined by detecting size differences in theamplification products, e.g., on an electrophoretic gel. For example,deletions or insertions of CFTR gene regions may be particularlyamenable to using size-based approaches.

Certain exemplary nucleic acid analysis methods are described in detailbelow.

Allele-Specific Amplification

In some embodiments, CFTR gene mutations are detected using anallele-specific amplification assay. This approach is variously referredto as PCR amplification of specific allele (PASA) (Sarkar, et al., 1990Anal. Biochem. 186:64-68), allele-specific amplification (ASA) (Okayama,et al., 1989 J. Lab. Clin. Med. 114:105-113), allele-specific PCR(ASPCR) (Wu, et al. 1989 Proc. Natl. Acad. Sci. USA. 86:2757-2760), andamplification-refractory mutation system (ARMS) (Newton, et al., 1989Nucleic Acids Res. 17:2503-2516). The entire contents of each of thesereferences is incorporated herein. This method is applicable for singlebase substitutions as well as micro deletions/insertions.

For example, for PCR-based amplification methods, amplification primersmay be designed such that they can distinguish between different alleles(e.g., between a wild-type allele and a mutant allele). Thus, thepresence or absence of amplification product can be used to determinewhether a CFTR gene mutation is present in a given nucleic acid sample.In some embodiments, allele specific primers can be designed such thatthe presence of amplification product is indicative of a CFTR genemutation. In some embodiments, allele specific primers can be designedsuch that the absence of amplification product is indicative of a CFTRgene mutation.

In some embodiments, two complementary reactions are used. One reactionemploys a primer specific for the wild type allele (“wild-type-specificreaction”) and the other reaction employs a primer for the mutant allele(“mutant-specific reaction”). The two reactions may employ a commonsecond primer. PCR primers specific for a particular allele (e.g., thewild-type allele or mutant allele) generally perfectly match one allelicvariant of the target, but are mismatched to other allelic variant(e.g., the mutant allele or wild-type allele). The mismatch may belocated at/near the 3′ end of the primer, leading to preferentialamplification of the perfectly matched allele. Whether an amplificationproduct can be detected from one or in both reactions indicates theabsence or presence of the mutant allele. Detection of an amplificationproduct only from the wild-type-specific reaction indicates presence ofthe wild-type allele only (e.g., homozygosity of the wild-type allele).Detection of an amplification product in the mutant-specific reactiononly indicates presence of the mutant allele only (e.g. homozygosity ofthe mutant allele). Detection of amplification products from bothreactions indicate (e.g., a heterozygote). As used herein, this approachwill be referred to as “allele specific amplification (ASA).”

Allele-specific amplification can also be used to detect duplications,insertions, or inversions by using a primer that hybridizes partiallyacross the junction. The extent of junction overlap can be varied toallow specific amplification.

Amplification products can be examined by methods known in the art,including by visualizing (e.g., with one or more dyes) bands of nucleicacids that have been migrated (e.g., by electrophoresis) through a gelto separate nucleic acids by size.

Allele-Specific Primer Extension

In some embodiments, an allele-specific primer extension (ASPE) approachis used to detect CFTR gene mutations. ASPE employs allele-specificprimers that can distinguish between alleles (e.g., between a mutantallele and a wild-type allele) in an extension reaction such that anextension product is obtained only in the presence of a particularallele (e.g., mutant allele or wild-type allele). Extension products maybe detectable or made detectable, e.g., by employing a labeleddeoxynucleotide in the extension reaction. Any of a variety of labelsare compatible for use in these methods, including, but not limited to,radioactive labels, fluorescent labels, chemiluminescent labels,enzymatic labels, etc. In some embodiments, a nucleotide is labeled withan entity that can then be bound (directly or indirectly) by adetectable label, e.g., a biotin molecule that can be bound bystreptavidin-conjugated fluorescent dyes. In some embodiments, reactionsare done in multiplex, e.g., using many allele-specific primers in thesame extension reaction.

In some embodiments, extension products are hybridized to a solid orsemi-solid support, such as beads, matrix, gel, among others. Forexample, the extension products may be tagged with a particular nucleicacid sequence (e.g., included as part of the allele-specific primer) andthe solid support may be attached to an “anti-tag” (e.g., a nucleic acidsequence complementary to the tag in the extension product). Extensionproducts can be captured and detected on the solid support. For example,beads may be sorted and detected. One such system that can be employedin this manner is the LUMINEX™ MAP system, which can be adapted forcystic fibrosis mutation detection by Luminex Corporation and is soldcommercially as a universal bead array (TAG-IT™) (See, e.g., Example 2)

Additional ASPE methods and reagents are described in, e.g., U.S. patentpublication number 2008/0138803 A1, the entire contents of which areherein incorporated by reference.

Single Nucleotide Primer Extension

In some embodiments, a single nucleotide primer extension (SNuPE) assayis used, in which the primer is designed to be extended by only onenucleotide. In such methods, the identity of the nucleotide justdownstream (e.g., 3′) of the 3′ end of the primer is known and differsin the mutant allele as compared to the wild-type allele. SNuPE can beperformed using an extension reaction in which the only one particularkind of deoxynucleotide is labeled (e.g., labeled dATP, labeled dCTP,labeled dGTP, or labeled dTTP). Thus, the presence of a detectableextension product can be used as an indication of the identity of thenucleotide at the position of interest (e.g., the position justdownstream of the 3′ end of the primer), and thus as an indication ofthe presence or absence of a mutation at that position. SNuPE can beperformed as described in U.S. Pat. No. 5,888,819; U.S. Pat. No.5,846,710; U.S. Pat. No. 6,280,947; U.S. Pat. No. 6,482,595; U.S. Pat.No. 6,503,718; U.S. Pat. No. 6,919,174; Piggee, C. et al. Journal ofChromatography A 781 (1997), p. 367-375 (“Capillary Electrophoresis forthe Detection of Known Point Mutations by Single-Nucleotide PrimerExtension and Laser-Induced Fluorescence Detection”); Hoogendoom, B. etal., Human Genetics (1999) 104:89-93, (“Genotyping Single NucleotidePolymorphism by Primer Extension and High Performance LiquidChromatography”), the entire contents of each of which are hereinincorporated by reference.

In some embodiments, primer extension can be combined with massspectrometry for accurate and fast detection of the presence or absenceof a mutation. See, U.S. Pat. No. 5,885,775 to Haff et al. (analysis ofsingle nucleotide polymorphism analysis by mass spectrometry); U.S. Pat.No. 7,501,251 to Koster (DNA diagnosis based on mass spectrometry); theteachings of both of which are incorporated herein by reference.Suitable mass spectrometric format includes, but is not limited to,Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF),Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), FourierTransform, and combinations thereof.

Oligonucleotide Ligation Assay

In some embodiments, an oligonucleotide ligation assay (“OLA” or “OL”)is used. OLA employs two oligonucleotides that are designed to becapable of hybridizing to abutting sequences of a single strand of atarget molecules. Typically, one of the oligonucleotides isbiotinylated, and the other is detectably labeled, e.g., with astreptavidin-conjugated fluorescent moiety. If the precise complementarysequence is found in a target molecule, the oligonucleotides willhybridize such that their termini abut, and create a ligation substratethat can be captured and detected. See e.g., Nickerson et al. (1990)Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927, Landegren, U. et al. (1988)Science 241:1077-1080 and U.S. Pat. No. 4,998,617, the entire contentsof which are herein incorporated by reference in their entirety.

Hybridization Approach

In some embodiments, nucleic acids are analyzed by hybridization usingone or more oligonucleotide probes specific for a region in the CFTRgene (SEQ ID NO:1) corresponding to the one or more mutations selectedfrom Table 1, 2, 3 or 4, and under conditions sufficiently stringent todisallow a single nucleotide mismatch. In certain embodiments, suitablenucleic acid probes can distinguish between a normal CFTR gene and amutant CFTR gene containing one or more mutations selected from Table 1,2, 3, or 4. For example, suitable nucleic acid probes specifically bindto a normal CFTR gene but not to a mutant CFTR gene containing one oremore mutations selected from Table 1, 2, 3, or 4. Alternatively, nucleicacid probes specifically bind to a mutant CFTR gene containing one ormore mutations selected from Table 1, 2, 3, or 4 but not to a normalCFTR gene. Probes of the present invention include those that arecapable of specifically hybridizing a mutant CFTR allele containing oneor more mutations listed in Tables 1, 2, 3, or 4. Probes of the presentinvention also include those that are capable of specificallyhybridizing a normal allele in a particular region of the CFTR gene andtherefore capable of distinguishing a normal allele from a mutant CFTRallele containing one or more mutations listed in Tables 1, 2, 3, or 4.Thus, for example, one of ordinary skill in the art could use probes ofthe invention to determine whether an individual is homozygous orheterozygous for a particular allele.

Nucleic acid hybridization techniques are well known in the art. See,e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Press, Plainview, N.Y. Those skilledin the art understand how to estimate and adjust the stringency ofhybridization conditions such that sequences having at least a desiredlevel of complementary will stably hybridize, while those having lowercomplementary will not. For examples of hybridization conditions andparameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview,N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in MolecularBiology. John Wiley & Sons, Secaucus, N.J.

In some embodiments, probe molecules that hybridize to the mutant orwildtype CFTR sequences can be used for detecting such sequences in theamplified product by solution phase or, more preferably, solid phasehybridization. Solid phase hybridization can be achieved, for example,by attaching the CFTR probes to a microchip.

Nucleic acid probes may comprise ribonucleic acids and/ordeoxyribonucleic acids. In some embodiments, provided nucleic acidprobes are oligonucleotides (i.e., “oligonucleotide probes”). Generally,oligonucleotide probes are long enough to bind specifically to ahomologous region of the CFTR gene, but short enough such that adifference of one nucleotide between the probe and the nucleic acidsample being tested disrupts hybridization. Typically, the sizes ofoligonucleotide probes vary from approximately 10 to 100 nucleotides. Insome embodiments, oligonucleotide probes vary from 15 to 90, 15 to 80,15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 35, 15 to 30, 18 to 30, or18 to 26 nucleotides in length. As appreciated by those of ordinaryskill in the art, the optimal length of an oligonucleotide probe maydepend on the particular methods and/or conditions in which theoligonucleotide probe may be employed.

In some embodiments, nucleic acid probes are useful as primers, e.g.,for nucleic acid amplification and/or extension reactions.

In some embodiments, nucleic acid probes are labeled with a detectablemoiety as described herein.

Arrays

A variety of the methods mentioned herein may be adapted for use witharrays that allow sets of mutations to be analyzed and/or detected in asingle experiment. For example, multiple novel CFTR mutations describedherein (e.g., Tables 1, 2, 3 or 4) can be analyzed at the same time.Additionally or alternatively, one or more novel CFTR mutationsdescribed herein (e.g., Tables 1, 2, 3 or 4) can be analyzed togetherwith other CFTR mutations known in the art at the same time. Inparticular, methods that involve use of nucleic acid reagents (e.g.,probes, primers, oligonucleotides, etc.) are particularly amenable foradaptation to an array-based platform (e.g., microarray). In someembodiments, an array containing one or more probes specific fordetecting CFTR mutations described herein (e.g., Tables 1, 2, 3 or 4)can be designed and adapted for various methods described herein.Additionally or alternatively, probes specific for detecting CFTRmutations described herein (e.g., Tables 1, 2, 3 or 4) can be combinedwith probes specific for CFTR mutations known in the art. In someembodiments, an array containing multiple probes are known as a mutationpanel. See, e.g., Wall et al. “A 31-mutation assay for cystic fibrosistesting in the clinical molecular diagnostics laboratory,” HumanMutation, 1995; 5(4):333-8, the entire contents of which are hereinincorporated by reference. Other methods may include the use ofreal-time PCR with probes for detecting CFTR mutations as describedherein.

Protein-Based Analyses

In certain embodiments, CFTR mutations are detected at the protein (orpeptide or polypeptide level), that is, a gene product from a CFTR genemutation is analyzed. For example, CFTR protein or fragment thereof canbe analyzed by amino acid sequencing methods, or immuno assays using oneor more antibodies that specifically recognize one or more epitopescorresponding to the one or more novel mutations described herein (e.g.,Table 1, 2, 3 and 4). CFTR proteins can also be analyzed by proteasedigestion (e.g., trypsin digestion) and, in some embodiments, thedigested protein products can be further analyzed by 2D-gelelectrophoresis.

Antibody Detection of Mutant Proteins

For example, specific antibodies that can differentiate between a normalCFTR protein and a mutant CFTR protein can be employed in any of avariety of methods known in the art to detect CFTR mutations. In certainembodiments, suitable antibodies can distinguish between a normal CFTRprotein and a mutant CFTR protein containing one or mutations selectedfrom Tables 1, 2, 3, or 4. For example, suitable antibodies specificallybind to a normal CFTR protein but not to a mutant CFTR proteincontaining one or more mutations selected from Table 1, 2, 3, or 4.Alternatively, suitable antibodies specifically bind to a mutant CFTRprotein containing one or more mutations selected from Table 1, 2, 3, or4 but not to a normal CFTR protein.

Antibodies against particular epitopes, polypeptides, and/or proteins(e.g., mutant or normal CFTR proteins) can be generated using any of avariety of known methods in the art. For example, the epitope,polypeptide, or protein against which an antibody is desired can beproduced and injected into an animal, typically a mammal (such as adonkey, mouse, rabbit, horse, chicken, etc.), and antibodies produced bythe animal can be collected from the animal. Monoclonal antibodies canalso be produced by generating hybridomas that express an antibody ofinterest with an immortal cell line. For more details on methods ofproducing, and uses of, antibodies to detect CFTR mutants, see, e.g.,U.S. Pat. No. 5,776,677, the entire contents of which are hereinincorporated by reference.

In some embodiments, antibodies are labeled with a detectable moiety asdescribed herein.

Antibody detection methods are well known in the art including, but arenot limited to, enzyme-linked immunosorbent assays (ELISAs) and Westernblots. Some such methods are amenable to being performed in an arrayformat. For example, a variety of different antibodies, each of which isspecific for different epitopes within the CFTR protein, could beimmobilized in an array and used in an assay such as an ELISA.

Detectable Moieties

In certain embodiments, certain molecules (e.g., nucleic acid probes,antibodies, etc.) used in accordance with and/or provided by theinvention comprise one or more detectable entities or moieties, i.e.,such molecules are “labeled” with such entities or moieties.

Any of a wide variety of detectable agents can be used in the practiceof the present invention. Suitable detectable agents include, but arenot limited to: various ligands, radionuclides; fluorescent dyes;chemiluminescent agents (such as, for example, acridinum esters,stabilized dioxetanes, and the like); bioluminescent agents; spectrallyresolvable inorganic fluorescent semiconductors nanocrystals (i.e.,quantum dots); microparticles; metal nanoparticles (e.g., gold, silver,copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes;colorimetric labels (such as, for example, dyes, colloidal gold, and thelike); biotin; dioxigenin; haptens; and proteins for which antisera ormonoclonal antibodies are available.

In some embodiments, the detectable moiety is biotin. Biotin can bebound to avidins (such as streptavidin), which are typically conjugated(directly or indirectly) to other moieties (e.g., fluorescent moieties)that are detectable themselves.

Below are described some non-limiting examples of other detectablemoieties.

Fluorescent Dyes

In certain embodiments, a detectable moiety is a fluorescent dye.Numerous known fluorescent dyes of a wide variety of chemical structuresand physical characteristics are suitable for use in the practice of thepresent invention. A fluorescent detectable moiety can be stimulated bya laser with the emitted light captured by a detector. The detector canbe a charge-coupled device (CCD) or a confocal microscope, which recordsits intensity.

Suitable fluorescent dyes include, but are not limited to, fluoresceinand fluorescein dyes (e.g., fluorescein isothiocyanine or FITC,naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein,6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryldyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes(e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G,rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.),coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes(e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.),Texas Red, Texas Red-X, SPECTRUM RED™, SPECTRUM GREEN™, cyanine dyes(e.g., CY-3™, CY-5™, CY-3.5™, CY5.5™, etc.), ALEXA FLUOR™ dyes (e.g.,ALEXA FLUOR™ 350, ALEXA FLUOR™ 488, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546,ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 633, ALEXA FLUOR™ 660,ALEXA FLUOR™ 680, etc.), BODIPY™ dyes (e.g., BODIPY™ FL, BODIPY™ R6G,BODIPY™ TMR, BODIPY™ TR, BODIPY™ 530/550, BODIPY™ 558/568, BODIPY™564/570, BODIPY™ 576/589, BODIPY™ 581/591, BODIPY™ 630/650, BODIPY™650/665, etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), and thelike. For more examples of suitable fluorescent dyes and methods forcoupling fluorescent dyes to other chemical entities such as proteinsand peptides, see, for example, “The Handbook of Fluorescent Probes andResearch Products”, 9th Ed., Molecular Probes, Inc., Eugene, Oreg.Favorable properties of fluorescent labeling agents include high molarabsorption coefficient, high fluorescence quantum yield, andphotostability. In some embodiments, labeling fluorophores exhibitabsorption and emission wavelengths in the visible (i.e., between 400and 750 nm) rather than in the ultraviolet range of the spectrum (i.e.,lower than 400 nm). For example, a suitable dye for use in real-time PCRprocedures may include SYBR Green.

A detectable moiety may include more than one chemical entity such as influorescent resonance energy transfer (FRET). Resonance transfer resultsan overall enhancement of the emission intensity. For instance, see Juet. al. (1995) Proc. Nat'l Acad. Sci. (USA) 92:4347, the entire contentsof which are herein incorporated by reference. To achieve resonanceenergy transfer, the first fluorescent molecule (the “donor” fluor)absorbs light and transfers it through the resonance of excitedelectrons to the second fluorescent molecule (the “acceptor” fluor). Inone approach, both the donor and acceptor dyes can be linked togetherand attached to the oligo primer. Methods to link donor and acceptordyes to a nucleic acid have been described previously, for example, inU.S. Pat. No. 5,945,526 to Lee et al., the entire contents of which areherein incorporated by reference. Donor/acceptor pairs of dyes that canbe used include, for example, fluorescein/tetramethylrohdamine,IAEDANS/fluroescein, EDANS/DABCYL, fluorescein/fluorescein, BODIPYFL/BODIPY FL, and Fluorescein/ QSY 7 dye. See, e.g., U.S. Pat. No.5,945,526 to Lee et al. Many of these dyes also are commerciallyavailable, for instance, from Molecular Probes Inc. (Eugene, Oreg.).Suitable donor fluorophores include 6-carboxyfluorescein (FAM),tetrachloro-6-carboxyfluorescein (TET),2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and thelike.

Enzymes

In certain embodiments, a detectable moiety is an enzyme. Examples ofsuitable enzymes include, but are not limited to, those used in anELISA, e.g., horseradish peroxidase, beta-galactosidase, luciferase,alkaline phosphatase, etc. Other examples include beta-glucuronidase,beta-D-glucosidase, urease, glucose oxidase, etc. An enzyme may beconjugated to a molecule using a linker group such as a carbodiimide, adiisocyanate, a glutaraldehyde, and the like.

Radioactive Isotopes

In certain embodiments, a detectable moiety is a radioactive isotope.For example, a molecule may be isotopically-labeled (i.e., may containone or more atoms that have been replaced by an atom having an atomicmass or mass number different from the atomic mass or mass numberusually found in nature) or an isotope may be attached to the molecule.Non-limiting examples of isotopes that can be incorporated intomolecules include isotopes of hydrogen, carbon, fluorine, phosphorous,copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium,bismuth, astatine, samarium, and lutetium (i.e., ³H, ¹³C, ¹⁴C, ¹⁸F, ¹⁹F,³²P, ³⁵S, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁹⁰Y, ^(99m)Tc, ¹¹¹In, ¹²⁵I, ¹²³I, ¹²⁹I,¹³¹I, ¹³⁵I, ¹⁸⁶Re, ¹⁸⁷Re, ²⁰¹Tl, ²¹²Bi, ²¹³Bi, ²¹¹At, ¹⁵³Sm, ¹⁷⁷Lu).

In some embodiments, signal amplification is achieved using labeleddendrimers as the detectable moiety (see, e.g., Physiol Genomics3:93-99, 2000), the entire contents of which are herein incorporated byreference in their entirety. Fluorescently labeled dendrimers areavailable from Genisphere (Montvale, N.J.). These may be chemicallyconjugated to the oligonucleotide primers by methods known in the art.

Kits

In certain embodiments, the invention provides kits for use inaccordance with the invention. Generally, inventive kits comprise one ormore reagents that differentiate a normal CFTR gene or protein from amutant CFTR gene or protein containing one or more mutations selectedfrom Table 1, 2, 3, or 4. For example, kits may comprise one or more(e.g., any combination of) reagents as described herein, and optionallyadditional components. For example, a kit according to the presentinvention may also include reagents that can detect other CFTR mutationswell known in the art.

Suitable reagents may include nucleic acid probes and/or antibodies orfragments thereof. In some embodiments, suitable reagents are providedin a form of an array such as a microarray or a CFTR mutation panel.

In some embodiments, provided kits further comprise reagents for carriedout various detection methods described herein (e.g., sequencing,hybridization, primer extension, multiplex ASPE, immuno assays, etc.).For example, kits according to the invention may optionally containbuffers, enzymes, and/or reagents for use in methods described herein,e.g., for amplifying nucleic acids via primer-directed amplification,for performing ELISA experiments, etc.

In some embodiments, provided kits further comprise a control indicativeof a healthy individual, e.g., a nucleic acid and/or protein sample froman individual who does not carry a CFTR mutation associated with CF or aCF related disorder. In some embodiments, provided kits further comprisea control indicative of known CFTR mutant alleles (such as ΔF508). Kitsmay also contain instructions on how to determine if an individual hasCF or a CF related disorder, is at risk of developing CF or a CF relateddisorder, or is a carrier of CFTR mutation.

In some embodiments, a computer readable medium encoding informationcorresponding to one or more mutations shown in Tables 1, 2, 3, and 4 isprovided. Such computer readable medium may be included in a kit of theinvention.

Systems

In an embodiment, the present invention provides systems for carryingout the analysis of the invention. Thus, in an embodiment, the presentinvention comprises a computer-readable medium on which is encodedprogramming code for the methods described herein. Also in anembodiment, present invention may comprise a system comprising aprocessor in communication with a computer-readable medium, theprocessor configured to perform the methods described herein. Suitableprocessors and computer-readable media for various embodiments of thepresent invention are described in greater detail below and areillustrated in FIG. 5.

Thus, in certain embodiments, the invention comprises a system forpredicting the activity of at least one gene comprising: a computerreadable medium; and a processor in communication with the computerreadable medium, the processor configured to estimate the effects ofindividual mutations in the at least one gene. The processor may, incertain embodiments, be further in communication with a databasecomprising data for a plurality of sequences for the portion of the atleast one gene, where the processor is configured to compare the nucleicacid and/or amino acid sequence of the portion of the at least one geneto the data of the plurality of sequences for the portion of the atleast one gene to determine if there is a mutation in the portion of theat least one gene in the biological sample obtained from the subject.

In other embodiments, the invention comprises a computer readable mediumon which is encoded program code for predicting the activity of at leastone gene, the program code comprising code for applying a model toestimate the effects of individual mutations in the at least one gene.In certain embodiments, the programming code comprises code configuredto compare the amino acid and/or nucleic acid sequence of the portion ofthe at least one gene to the data for a plurality of sequences for theportion of the at least one gene stored in a database to determine ifthere is a mutation in the portion of the at least one gene in thebiological sample obtained from the subject.

Some embodiments of the systems and computer readable media of theinvention may be applied to various genes. In certain embodiments, theat least one gene comprises the CFTR gene.

As noted herein, the sequence of the portion of the at least one geneand the biological activity of interest as assessed for a particularsubject may be compared to a database of amino acid and/or nucleic acidsequences and biological activity as assess for a plurality of subjects.Thus, in certain embodiments of the systems and computer readable media,the database comprises data for the biological activity as measured in aplurality of samples from which the sequence of the portion of the atleast one gene was determined.

Embodiments in accordance with aspects of the present subject matter canbe implemented in digital electronic circuitry, in computer hardware,firmware, software, or in combinations of the preceding. In oneembodiment, a computer may comprise a processor or processors. Theprocessor may comprise, or have access to, a computer-readable medium,such as a random access memory coupled to the processor. The processormay execute computer-executable program instructions stored in memory,such as executing one or more computer programs including a samplingroutine and suitable programming to produce output to generate theanalysis described in detail herein.

Such processors may comprise a microprocessor, a digital signalprocessor (DSP), an application-specific integrated circuit (ASIC),field programmable gate arrays (FPGAs), and state machines. Suchprocessors may further comprise programmable electronic devices such asPLCs, programmable interrupt controllers (PICs), programmable logicdevices (PLDs), programmable read-only memories (PROMs), electronicallyprogrammable read-only memories (EPROMs or EEPROMs), or other similardevices.

Such processors may comprise, or may be in communication with, media,for example tangible computer-readable media that may store instructionsthat when executed by the processor, can cause the processor to performthe steps described herein as carried out, or assisted, by a processor.Embodiments of computer-readable media may comprise, but are not limitedto, all electronic, optical, magnetic, or other storage devices capableof providing a processor, such as the processor in a web server, withcomputer-readable instructions. Other examples of media comprise, butare not limited to, a floppy disk, CD-ROM, magnetic disk, memory chip,ROM, RAM, ASIC, configured processor, all optical media, all magnetictape or other magnetic media, or any other medium from which a computerprocessor can read. Also, various other devices may includecomputer-readable media, such as a router, private or public network, orother transmission device. The processor, and the processing may be inone or more structures, and may be dispersed through one or morestructures. The processor may comprise code for carrying out one or moreof the methods (or parts of methods) described herein.

The system may comprise a data compiling system as well as a means forthe user to interact with the system as the analysis proceeds. Thus, inan embodiment, the present invention may comprise a system forcollecting and/or compiling data from a plurality of assay measurementsand/or sequencing data and transmitting the data to a computer, and asystem for transmitting the results of the analysis to a user. Thesystems of the present invention may be designed for high-throughputanalysis of DNA and/or amino acid sequencing data. Thus, in anembodiment, the plurality of measured signals comprise a plurality ofknown DNA sequences isolated from at least one cell type.

FIG. 5 shows an embodiment of the flow of information in a systemcomprising the software of the present invention. As discussed above, acomputer processor or CPU may include, for example, digital logicprocessors capable of processing input, executing algorithms, andgenerating output as necessary in response to the inputs received fromthe touch-sensitive input device. As detailed herein, such processorsmay include a microprocessor, such as an ASIC, and state machines,and/or other components. Such processors include, or may be incommunication with, media, for example computer-readable media, whichstores instructions that, when executed by the processor, cause theprocessor to perform the steps described herein.

Thus, in an embodiment, the starting point may comprise data (100) thatmay comprise a normal CFTR gene (100A) and mutant CFTR gene (100B). Oncethe data has been collected (110), it may be compiled (120) and/ortransformed if necessary using any standard spreadsheet software such asMicrosoft Excel, FoxPro, Lotus, or the like. In an embodiment, the dataare entered into the system for each experiment. Alternatively, datafrom previous runs are stored in the computer memory (150) and used asrequired.

At each point in the analysis, the user may input instructions via akeyboard (180), floppy disk, remote access (e.g., via the internet)(190), or other access means. The user may enter instructions includingoptions for the run, how reports should be printed out, and the like.Also, at each step in the analysis, the data may be stored in thecomputer using a storage device common in the art such as disks, drivesor memory (150). As is understood in the art, the processor (160) andI/O controller (170) are required for multiple aspects of computerfunction. Also, in a embodiment, there may be more than one processor.

The data may also be processed to remove noise (130). In some cases, theuser, via the keyboard (180), floppy disk, or remote access (190), maywant to input variables or constraints for the analysis, as for example,the threshold for determining noise. The results of the analysis maythen be compiled and provided in a form for review by a user (140).

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

EXAMPLES Example 1 Identification of Novel Mutations in the CFTR Gene

Novel mutations in the CFTR gene were identified using the CF fullsequencing assay. Typically, samples submitted for CF full sequencingassays were from individuals for whom testing in CF mutation panels hasbeen uninformative, or partially uninformative. These individualsinclude 1) patients with idiopathic chronic pancreatitis; 2) patientswith congenital bilateral absence of the vas deferens (CBAVD); 3)couples who test positive/negative by mutation analysis; 4) CF-affectedor suspected patients in whom one or no mutations have been identified;5) obligate carriers of a rare familial mutation; 6) patients with afamily history of CF, for whom mutation analysis by other methodologiesis negative; 7) patients with a CF related disease or condition.Sequence changes in the CFTR gene were identified by comparing thepatient gene sequence to the wild-type gene sequence. Novel mutations inthe CFTR gene that were unreported previously are summarized in Table 5.

As shown in Table 5, patients carrying these novel mutations were fromdifferent ethnic groups including Caucasians, African Americans,Hispanics, and Asians. Some of the mutations are located in introns(e.g., intron 3, intron 6a, intron 11, intron 14a, intron 19, intron 20,intron 21, and intron 23). Some of the mutations are located in exons(e.g., exon 2, exon 3, exon 4, exon 5, exon 6a, exon 6b, exon 7, exon 9,exon 10, exon 11, exon 12, exon 13, exon 14a, exon 14b, exon 15, exon16, exon 17a, exon 17b, exon 19, exon 20, exon 21, exon 22, and exon24).

As shown in Table 5, most of the novel mutations identified result incodon changes or altered gene splicing sites, which will likely affectthe CFTR gene expression and/or protein function. In particular, some ofthe mutations are nonsense mutations (i.e., mutations predicted toresult in the introduction of a stop codon). Some of the mutationsaffect consensus splice site ag/gt. Some of these mutations areinsertion or deletion of at least one nucleotide. These mutations arecategory 2 mutations according to the ACMG guidelines, and are of thetype expected to cause CF or CF related disease, disorder or condition.

Some mutations are missense mutations. Some are predicted to cause causein-frame insertions and/or deletions. Some are likely to affect splicesites. These mutations are category 3 mutations according to the ACMGguidelines.

Thus, the novel mutations provided herein can be used, alone or incombination with other known CF mutations, to detect CF or a CF relateddisorder in CFTR testing assays including carrier testing.

TABLE 5 Novel Mutations in the CFTR gene Sequence Type of AA OtherChange mutation Change Ethnicity Mutations Clinical Information Exon1824delA Frame- n/a Caucasian F508delMutation was identified in a 22 year old e12 shiftpatient with a known diagnosis of CF. (FS)This patient carried a second mutation known to cause CF (F508del).2957delT FS n/a Caucasian F508delMutation was identified in a 1 year old e15patient with a known diagnosis of CF.The patient carried a second mutation known to cause CF (F508del).4089ins4 FS n/a Caucasian F508delMutation was identified in a 7 year old e21patient with a known diagnosis of CF.The patient had a positive sweat chloridetest. The patient carried a second mutation known to cause CF (F508del).4374 + Splice n/a 1.  1. F508delPatient #1: Mutation was identified in a i23 2T > C site Caucasian45 year old patient with a suspected mutation 2.  2. F508deldiagnosis of CF. The patient carried a Caucasiansecond mutation known to cause CF (F508del).Patient #2: Mutation was identified in a52 year old patient with a suspecteddiagnosis of CF. The patient carried a second mutation known to cause CF(F508del). 3064A > T Nonsense K978X African Q1042XMutation was identified in a 26 year old e16 Americanpatient with a known diagnosis of CF.The patient carried a second mutation likely to cause CF Q1042X. 246C >G Nonsense Y38X Caucasian F508delMutation was identified in a 1 month old e2patient with a suspected diagnosis of CF.The patient had a positive sweat chloridetest. The patient carried a second mutation known to cause CF (F508del).269C > T Missense A46V 1)  1) 3849 +Patient #1: Mutation was identified in a e2 (MS) Caucasian 12192G > A32 year old patient who was tested due to 2) Black 2) F508delabnormalities found on fetal ultrasound. 3) African 3) noneThe patient carried a second mutation of Americanunknown clinical significance (3849 + 12192G > A).Patient #2: Mutation was identified in a 2month old patient who was tested basedon follow-up for a positive newborn screen. The patient carried a secondmutation known to cause cystic fibrosis (F508del).Patient #3: Mutation was identified in a24 year old patient who was tested as aparental follow-up to a positive newborn screen. 2902 G > T MS D924YCaucasian F508del Mutation was identified in a 1 month old e15patient with a suspected diagnosis of CF.The patient also had a positive sweat chloride test and carried a secondmutation known to cause CF (F508del) 3814G > A MS E1228K CaucasianF508del Mutation was identified in a 1 month old e19patient with a suspected diagnosis of CF.The patient had a borderline sweat chloride test. The patient carried asecond mutation known to cause CF (F508del). 502G > C MS G124R NotF508del Mutation was identified in a 2 month old e4 Providedpatient with a suspected diagnosis of CF.The patient had a positive sweat chloridetest. The patient carried a second mutation known to cause CF (F508del).1520G > T MS G463V Caucasian F508delMutation was identified in a 17 year old e9patient with a known diagnosis of CF.Patient carried a second mutation known to cause CF (F508del). 511_513In frame L127dup Caucasian, W1282X Mutation was identified in a newborne4 dup TTA dupli- Asian with a suspected diagnosis of CF. The cationpatient had clinical symptoms of CFincluding as a positive sweat chloridetest, meconium ileus, echogenic bowel,and pancreatic insufficiency. The patientcarried a second mutation known to cause CF (W1282X). 978A > T MS E282D1. not 1. Patient #1: Mutation was identified in a e6b provided 3120 +10 year old patient with a suspected 1G > Adiagnosis of CF. The patient had a 2) not 2. nonepositive sweat chloride test. The patient providedcarried a second mutation known to cause CF (3120 + 1G > A).Patient #2: Mutation was identified in a 4year old patient with a suspecteddiagnosis of CF and a family history of CF. 843G > C MS Q237H CaucasianF508del Mutation was identified in a 2 month old e6apatient with a known diagnosis of CF.The patient carried a second mutation known to cause CF (F508del).829C > T MS L233F Caucasian D1152HMutation was identified in a 1 month old e6apatient who was tested following a positive newborn screen. The patientcarried a second mutation known to cause CF (D1152H). 4096-6C > T SpliceNone Caucasian F508del Mutation was identified in a 58 year old i21 sitepatient with a suspected diagnosis of CF. mutationThe patient carried a second mutation known to cause CF (F508del).4375-7delT Splice None Caucasian F508delMutation was identified in a 6 year old i23 sitepatient with a suspected diagnosis of CF. mutationPatient has a family history, a borderlinesweat chloride test and recurrentpneumonia. The patient carried a secondmutation known to cause CF (F508del). 1586 G > C MS S485T CaucasianS1235R Mutation was identified in a 2 year old e10patient with a suspected diagnosis of CF.The patient carried a second mutation S1235R (3837T > G) which has beenreported in individuals with varying CF phenotypes. 875 + G > T Splicen/a African none Mutation was identified in a 1 month old i6a siteAmerican patient who had a positive newborn mutation screening test.4005 + Splice n/a Caucasian noneMutation was identified in a 40 year old i20 3G > T sitepatient who was tested to determine if mutationthey were a carrier, there was no family history of CF. 2711T > C MS1860T Caucasian F508del, Mutation was identified in a 58 year old e14aE528E woman with a suspected diagnosis of CF.The patient carried a second mutation known to cause CF (F508del) and anadditional mutation of unknown clinical significance (E528E).. 3891G > CMS L1253F Not G85E, L15P Mutation was identified in a 32 year old e20provided patient with a known diagnosis of CF.The patient carried a second mutation known to cause CF (G85E) and anadditional mutation of unknown clinical significance (L15P). 2524C > TMS P798S African F508del, Mutation was identified in a 5 year old e13American R74W, patient with a suspected diagnosis of CF. G921E,The patient had a positive sweat chloride D1270Ntest. This patient carried a second mutation known to cause CF (F508del)and three additional mutations of unknown clinical significance (R74W,G921E, D1270N). 2894G > A MS G921E African F508del,Mutation was identified in a 5 year old e15 American R74W,patient with a suspected diagnosis of CF. P798S,The patient had a positive sweat chloride D1270Ntest. This patient carried a second mutation known to cause CF (F508del)and three additional mutations of unknown clinical significance (R74W,P789S, D1270N). 405 + Possible n/a Caucasian F508delMutation was identified in a 35 year old i3 10247C  > T splice patient who was tested to determine if sitethey were a carrier, there was no family mutationhistory of CF. This patient carried a second mutation known to cause CF(F508del). 405 + 10255 Possible n/a Not F508del,Mutation was identified in a 10 year old i3 d elC splice  Provided124del23bp patient. The patient carries two mutations siteknow to cause CF (F508del and mutation 124del23). 1811 + Possible n/a1.  1. F508del Patient #1: Mutation was identified in a 1 i11 1643G > Tsplice  Hispanic year old patient with a known diagnosis site 2. 2. F508del of CF. Patient had a positive sweat mutation Hispanicchloride test. The patient carried a 3. Not 3. nonesecond mutation known to cause CF provided (F508del).Patient #2: Mutation was identified in a 6year old patient with a known diagnosisof CF. The patient carried a second mutation know to cause CF (F508del).Patient #3: Mutation was identified in an8 month old patient with a suspected diagnosis of CF. 1812- Splice n/aCaucasian none Mutation was identified in a 15 year old i11 13A > G sitepatient with a suspected diagnosis of CF. mutationThe patient has chronic sinusitis. 2752- Possible n/a African F693LMutation was identified in a 6 year old i14a 33insA splice  Americanpatient with a known diagnosis of CF. siteThe patient carries a second mutation of mutationunknown clinical significance (F693L). 3849 + Possible n/a CaucasianA46V Mutation was identified in a 32 year old i19 12192G > A splice patient who was tested due to siteabnormalities found on fetal ultrasound. mutationThe patient carried an additionalmutation of known clinical significance (A46V). 724G > A MS A198THispanic none Mutation was identified in a 4 month old e6apatient with a suspected diagnosis of CF. 3899C > T MS A1256V Guyanesenone Mutation was identified in a 45 year old e20patient who was tested to determine ifthey were a carrier, there was no family history of CF. 3986C > T MSA1285V Not none Mutation was identified in a 23 year old e20 Providedpatient who was tested to determine ifthey were a carrier, there was no family history of CF. 901G> A MS E257KHispanic none Mutation was identified in a 4 year old e6bpatient with a suspected diagnosis of CF.The patient has asthma and recurring pneumonia. 392 T > C MS F875 Notnone The mutation was identified in a 1 month e3 Providedold patient with a suspected diagnosis of CF. 3463T > C MS F1111LHispanic none Mutation was identified in a 6 year old e17bpatient with a suspected diagnosis of CF. The patient has asthma.1757G > A MS G542E Hispanic noneMutation was identified in a 25 year old e11patient who was tested to determine ifthey were a carrier, there was no familyhistory of CF. The patient carried 2 copies of G542E.. 4025G > C MSG1298A Asian G970D, Mutation was identified in a 34 year old e21 Q1352Hpatient with congenital absence of thevas deferens. The patient carried twoother mutations of unknown clinical significance (G970D and Q1352H)4129G > T MS G1333W Not none Mutation was identified in an 8 year olde22 Provided patient with a suspected diagnosis of CF.Patient had recurrent respiratory infections and chronic cough. 663T > GMS I177M Caucasian none Mutation was identified in a 34 year old e5patient who was tested to determine ifthey were a carrier, there was no family history of CF. 3200T > C MSI1023T Hispanic none Mutation was identified in a 34 year old e17apatient who was tested to determine ifthey were a carrier, there was no family history of CF. 4412T > C MSI1427T Asian S1444S Mutation was identified in a 34 year old e24patient who was tested to determine ifthey were a carrier, there was no familyhistory of CF. The patient carried anothermutation that is considered likely to be clinically benign (S144S).620A > C MS K163T Caucasian noneMutation was identified in a 32 year old e4patient with a family history of CF.

Example 2 CFTR Mutation Detection Assay

The present example demonstrates that multiplex ASPE assay can be usedto detect novel cystic fibrosis mutations described herein. MultiplexASPE combines multiplex PCR and allele-specific primer extension.Multiplex PCR is performed to amplify target regions in the CFTR genecontaining novel sequence variations described herein from genomic DNAin a sample. Multiplex primer extension reactions are then performedusing allele-specific primers, i.e., extension primers that possess a 3′terminal nucleotide, which form a perfect complement with the targetsequence, are extended to form extension products and modifiednucleotides (e.g., biotinylated dCTP) are incorporated into theextension product for detection purposes. Alternatively, an extensionprimer may instead contain a 3′ terminal nucleotide which forms amismatch with the target sequence. In this instance, primer extensiondoes not occur. Primer extension products are then hybridized touniversal array beads with “anti-tag” sequence (sequences complementaryto the tag sequence) for capture and detection purposes.

In some cases, the novel mutations described herein can be detected incombination of other known CF mutations, for example, mutationsrecommended by the American College of Genetics and American College ofObstetricians and Gynecologists, as well as other common and clinicallyrelevant mutations, such as, for example, AF508 (exon 10), G542X (exon11), G551D (exon 11), R117H (exon 4), W1282X (exon 20), N1303K (exon21), 3905insT (exon 20), 3849+10KbC>T (intron 19), G85E (exon 3), R334W(exon 7), A455E (exon 9), 1898+1G>A (exon 12), and/or 2184delA (exon13).

Various ASPE kits can be used to carry out the detection methodsdescribed herein. For example, Luminex's TAG-IT™ kit and Data Analysissoftware can be modified to detect a panel of CF mutations including oneor more novel mutations described herein. Mutation detection kit may usenon-isotopic fluorescent technology, and a 96-well assay format that iscompatible with automation such that result analyses and genotypecalling are automated.

Allele Specific Primers

Allele specific primers can be designed based on the sequence variationsshown in Table 5 and the CFTR genomic sequences (including exon andintron sequences) using various methods and software known in the art. Auniversal tag sequence can be added to allele specific primers.

Specimens and Assay Format

Specimens containing genomic DNA to be analyzed can be obtained from,but not limited to, the following sources: Whole blood (e.g., wholeblood in EDTA, ACD-A, ACD-B), fresh or frozen tissue, amniotic fluid,CVS (chorionic villus sampling) tissue, cultured cells (e.g., CVS,amniotic fluid, fibroblasts, POC (product of conception)), blood spots,cord blood, mouthwash, genomic DNA extracted by an outside laboratory.Blood and bloodspot DNA samples are typically run undiluted at a 5 μLinput volume. An amount of 5 to 200 ng DNA is used as input. For testingprenatal and mouthwash samples, generally between 20 ng and 150 ng isused as input, for example, about 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 45ng, 50 ng, 55 ng, 60 ng, 65 ng, 70 ng, 75 ng, 80 ng, 85 ng, 90 ng, 95ng, 100 ng, 110 ng, 120 ng, 130 ng, 140 ng, or 150 ng.

A 96-well assay plate is used. Two genomic DNA controls are includedwith each assay plate. The specific controls are rotated sequentiallythrough assay plates. Each assay plate also includes two cocktail blanksand ASPE (Allele-Specific Primer Extension) controls. A calibrating96-well filter plate is also used during data acquisition.

Single-Well Multiplex PCR

Multiplex PCR are performed to amplify exons containing mutationsdescribed herein using consensus flanking intron sequences. Generally,amplicons range in size between about 150 bp and 600 bp (inclusive ofendpoints).

Typically, 5 ng-200 ng of DNA is amplified to produce a productcontaining multiple amplicons using PCR amplification conditions knownin the art or optimized/modified using routine experimentation.

Enzymatic Post-PCR Cleanup

PCR products are treated with Exonuclease I and Shrimp AlkalinePhosphatase to remove residual primers that will interfere withallele-specific primer extension reactions. PCR products are incubatedwith enzyme and then enzyme is heat-deactivated, according to standardprotocols or modified protocols readily developed by one of ordinaryskill in the art.

Single Well Allele Specific Primer Extension (ASPE) Reactions

Typically up to 100 sequence variations can be distinguished in asingle-well reaction; using the Luminex bead set. For example, a set ofallele-specific oligonucleotide (ASO) primers (including wildtypecontrol ASOs) with tag sequences are used.

The Exo-SAP-treated PCR product is subjected to an allele-specificprimer extension reaction containing tagged primers and biotinylateddCTP using PCR reaction conditions known in the art or modified readilyby one of ordinary skill in the art.

Universal Array Sorting and Detection

Each bead is coupled with an anti-tag sequence complementary to the tagsequence ASPE primers. Therefore, any ASPE products, if present, can becaptured for genotype analysis. Wild-type control for each amplicon isincluded. The signals from wildtype alleles serve as a control for eachamplicon and provide information for allelic ratio calculation(typically obtained by calculating the ratio of signal for the mutantallele over signal for the wildtype allele), for the detected mutations.

The ASPE product is added to the universal bead array containinganti-tags to the ASPE primers and incubated for hybridization.Hybridization reactions are then washed over a filter that captures thebeads and removes any non-hybridized ASPE products containing biotin.Bead hybridization conditions are known in the art and can be adaptedreadily by one skilled in the art.

Strepatavidin R-Phycoerythrin conjugate is added to the hybridizedproducts on the filter plate and incubated at room temperature, followedby bead sorting and detection. For example, a modified LUMINEX™ 100 IS™or 200 IS™ can be used. The LUMINEX™ 100 IS™ can upload sample sheetsfrom text files or barcodes. Detection time averages 20-100 seconds perwell.

Results

In the LUMINEX™ system, results are generated as a <.csv> file andexported in batches. The batch output file (.csv) is opened in TAG-IT™Data Analysis Software (TDAS) version 6.0 where results areautomatically generated based on pre-determined algorithms for allelicratios on certain individually tested mutations and the presence orabsence of signal on the remaining mutations.

Mutation Confirmation

Samples positive for any of the mutations described herein can beconfirmed by a second assay run. Positive samples can also be confirmedby direct DNA sequencing.

Example 3 Cystic Fibrosis Sequencing Assays

The Cystic Fibrosis full sequencing assay and single exon sequence assaycan be used to detect mutations in the CFTR gene directly in a patientsample. The Cystic Fibrosis full sequencing assay and single exonsequence assay can also be used to complement CF screening panels,and/or to serve as a confirmatory assay for samples that are positivefor multiplex mutations or those without a normal counterpart in the CFmutation detection assay.

The CF full sequencing assay sequences the entire coding region of theCFTR gene plus 15 bp at the 3′ end of each intron (30 bp for e17b tocover a known mutation) and 6 bp at the 5′ beginning of each intron.

In addition, the assay includes portions of introns 1, 3, 11, and 19useful in identifying the exon 2, 3 deletion, the A>G mutation at1811+1.6 kb, and the C>T mutation at 3849+10 kb. Typically, the assaycomprises analysis of 31 amplicons: e1, i1, e2, e3, i3, e4, e5, e6a,e6b, e7, e8, e9, e10, e11, e12, e13a, e14a, e14b, e15, e16, e17a, e17b,e18, e19, i19, e20, e21, e22, e23, and e24. Each amplicon includes thecomplete coding region of the exon with the exception of 13.1 and 13.2,in which, due to the large size of the exon, the amplicon is dividedinto two fragments. The CF Single Exon Sequencing assay uses the sameprimers but on an individual basis as needed.

Samples tested in the CF single exon sequencing assay in this Exampleinclude those from individuals that 1) tested positive in a CF mutationdetection assay (e.g., multiplex ASPE assay as described in Examples 2)but require confirmation; 2) tested positive in the CF full sequencingassay and require repeat testing; 3) are being tested for a knownfamilial mutation(s); and/or 4) are being tested for a mutation that isnot detectable in the CF mutation detection assay of Example 2.

Specimens and Assay Format

Specimens to be analyzed can be extracted genomic DNA from any of, butnot limited to, the following sources: Whole blood (e.g., whole blood inEDTA, ACD-A, ACD-B), blood spots, amniotic fluid, chorionic villussamples (CVS) (for single exon sequencing only), cultured cells (e.g.,CVS, amniotic fluid, fibroblasts, POC), mouthwash (for single exonsequencing only).

A 96-well format is used. Cocktail blanks are run for all amplicons oneach assay.

PCR Amplification

Target regions containing mutations described herein are first amplifiedby PCR amplification. Typically, 5 ng-200 ng of DNA is amplified in a 25μL volume reaction. PCR primers include 5′ UPS tags-UPS1 for the Forwardprimers and UPS2 for the Reverse primers. Table 6 presents sequences ofexemplary primers used in amplification of certain exemplary target exonor intron regions.

TABLE 6 Primer sequences Amplicon SEQ ID Amplicon Primer NameSequence (5′-3′) Length NO. Primers for exonic sequences CF exon 1UP1CFe1F TTTAACCTGGGCAGTGAAG 373  5 UP2CFe1R AACCCAACCCATACACA  6CF exon 2 UP1CFe2F CAAATCAAGTGAATATCTGTTC 316  7 UP2CFe2RAGCCACCATACTTGGCTCCTA  8 CF exon 3 UP1CFe3F2 CTAAAATATTTGCACATGCAAC 333 9 UP2CFe3R TTTCTTAGTGTTTGGAGTTGG 10 CF exon 4 UP1CFe4F2TCATTTTAAGTCTCCTCTAAAG 407 11 UP2CFe4R CGATACAGAATATATGTGCCA 12CF exon 5 UP1CFe5F2 AACAACTAGAAGCATGCCAG 394 13 UP2CFe5R2GTTGTATAATTTATAACAATAGTG 14 CF exon 6a UP1CFe6aF2 GGAAGATACAATGACACCTG353 15 UP2CFe6aR3 CTGAAGATCACTGTTCTATGC 16 CF exon 6b UP1CFe6bF3ATGACTTAAAACCTTGAGCAGT 336 17 UP2CFe6bR2 GGAAGTCTACCATGATAAACAT 18CF exon 7 UP1CFe7F2 GAGACCATGCTCAGATCTTCC 507 19 UP2CFe7RACTTTTATAACTTCCTAGTGAAG 20 CF exon 8 UP1CFe8F2 AAGATGTAGCACAATGAGAGTA268 21 UP2CFe8R CAGTTAGGTGTTTAGAGCAA 22 CF exon 9 UP1CFe9FGTATACAGTGTAATGGATCATG 402 23 UP2CFe9R4 CACCAAATTAAGTTCTTAATAG 24CF exon 10 UP1CFe10F TTCTGCTTAGGATGATAATTGG 479 25 UP2CFe10RGCATAGGTCATGTGTTTTATTA 26 CF exon 11 UP1CFe11F CAGATTGAGCATACTAAAAGTG240 27 UTP2CFe11R TACATGAATGACATTTACAGCA 28 CF exon 12 UP1CFel2FGCTACTTCTGCACCACTTTTG 344 29 UP2CFe12R CAGTCTGTCTTTCTTTTATTTTA 30CF exon UP1CFe13F3 CAAAATGCTAAAATACGAGAC 388 31 13a UP2CFe13R5TCCAGGAGACAGGAGCATC 32 CF exon UP1CFe13F4 CTCATGGGATGTGATTCTTT 714 3313b UP2CFe13R2 GATACACCTTATCCTAATCCTA 34 CF exon UP1CFe14aF3ACCACAATGGTGGCATGA 299 35 14a UP2CFe14aR2 TGTATACATCCCCAAACTATC 36CF exon UP1CFe14bF TGGGCATGGGAGGAATAGGTG 228 37 14b UP2CFe14bRTTACAATACATACAAACATAGTG 38 G CF exon 15 UP1CFe15F2 AAGTAACTTTGGCTGC 41639 UP2CFe15R2 CTGCCATTAGAAAACCA 40 CF exon 16 UP1CFe16F2AAGTCTATCTGATTCTATTTGC 307 41 UP2CFe16R2 GTTTTTTTAATAATACAGACATAC 42 TCF exon 3 UP1CFe17aF TGTCCACTITGCAATGTGAA 317 43 17a UP2CFe17aR3CAATAAAGAATCTCAAATAGCTC 44 T CF exon 3 UP1CFe17bF TAGTCTTTTTCAGGTACAAG516 45 17b UP2CFe17bR6 CAATGGAAATTCAAAGAAATCAC 46 T CF exon 18UP1CFe18F6 GAATACTTACTATATGCAGAGCA 416 47 UP2CFe18R3GTTCTTCCTCATGCTATTACTC 48 CF exon 19 UP1CFe19F GCCCGACAAATAACCAAGTGA 49449 UP2CFe19R2 CTAACACATTGCTTCAGGCTA 50 CF exon 20 UP1CFe20FAAGGTTGTTTGTCTCCATATAT 544 51 UP2CFe20R GCCTATGAGAAAACTGCACT 52CF exon 21 UP1CFe21F ACATGGGTGTTTCTTATTTA 428 53 UP2CFe21R2GTTAGGGGTAGGTCCAGT 54 CF exon 22 UP1CFe22F GCTTGAGTGTTTTTAACTCTGTG 31455 UP2CFe22R ATGATTCTGTTCCCACTGTGC 56 CF exon 23 UP1CFe23FGTTCTGTGATATTATGTGTGG 226 57 UP2CFe23R CAAGGGCAATGAGATCTTAAG 58CF exon 24 UP1CFe24F2 AGTTTCTGTCCCTGCTCT 356 59 UP2CFe24RGAGCAAATGTCCCATGTCAAC 60 Primers for intronic sequences CF intron 1UP1CFin1F2A AATGGTGTTTACCTACCTAGAGA 250 61 UP4CFin1R2CCTCCTCTGATTCCACAAG 62 CF intron 3 UP3CFin3F3 CTGAGATTCTGTTCTAGGTGTG 36663 UP2CFin3R CCTACACTCAGAACCCATCAT 64 CF intron UP1CFin19FTTCAGTTGACTTGTCATCTTG 223 65 19 UP2CFin19R AATATGTTGAAAGTTAAACAGTG 66CF intron UP1CFin11F GTTACACTATAAAGGTTGTTTTAG 292 67 11 AC UP2CFin11RCACAGTTCCCATATTAATAGAAAT 68 G (Seq) CFe9.SEQF TTTTTAACAGGGATTTGGG N/A 69(Seq) CFe6bF2 GATTGATTGATTGATTGATT N/A 70 (Seq) UPS1GCGGTCGCATAAGGGTCAGT N/A 71 (Seq) UPS2 CGCCAGCGTATTCCCAGTCA N/A 72PCR conditions are as shown in Table 7.

TABLE 7 PCR amplification conditions for CF full sequencing assay CyclesTemperature (° C.) Time Function 1 95 5 min Denaturation of enzyme 35 9520 sec Denaturation of dsDNA 55 20 sec Annealing 72 40 sec Extension 172 7 min Final extension 1 8 Forever EndEnzymatic Post-PCR Clean Up

PCR products are treated with Exonuclease I (Exo) and Shrimp AlkalinePhosphatase (SAP) to remove residual primers that may interfere withsequencing. The following incubation conditions are used:

-   -   37° C. for 30 minutes (enzyme digestion)    -   99° C. for 15 minutes (enzyme deactivation)    -   Hold at 8° C. until storage    -   Products can be stored, e.g., at −80° C. or −20° C.        Sequencing

Exo-SAP treated products are diluted 1:2 in water, and 3 μL is added to7 μL of each forward and reverse sequence cocktail containing Big Dyev3.1 (ABI). In order to obtain bidirectional sequencing results, twosequencing reactions are performed for each amplicon, using both UPS1and UPS2 primers. An additional forward sequencing reaction using genespecific primers is performed for Exons 6b and 9 to obtain readablesequence beyond the repeat regions. Cycle sequencing is performed in athermocycler with the conditions shown in Table 8.

TABLE 8 Thermocycler conditions for sequencing reactions for CF fullsequencing assay Cycles Temperature (° C.) Time Function 1 96 1 minDenaturation of enzyme 25 96 10 sec Denaturation of dsDNA 53 5 secAnnealing 60 3 sec Extension 1 8 Forever End

Assay plates can be stored, e.g., at −80° C. for up to 2 weeks untilanalyzed or further manipulated.

Post-Sequencing Purification

Sequence products are purified using the Performa DTR Ultra 96 WellPlate (Edge Biosystems). Sequencing reactions are diluted 1:2 and 10 μLis purified through the Edge Plate.

Sequencing Run: ABI 3730 Genetic Analyzer and Data Analysis

A 1 kV/14 second injection is performed on the 3730×1 Genetic Analyzer.POP7 polymer and a 50 cm array are used for optimal resolution.Parameters for a typical sequencing run are shown in Table 9.

TABLE 9 Parameters for typical sequencing runs Feature Parameter for CFfull sequencing Run Temp 60° C. Pre Run Voltage 15.0 Kvolts Pre Run Time180 sec Injection Voltage 1.0 Kvolts Injection Time 14 sec Voltagenumber of steps 30 Voltage Step Interval 15 sec Data Delay Time 240 secRun Voltage 13.4 Kvolts Run Time 2400 sec

Sequence data Analysis is performed using SEQSCAPE™ software (ABI).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims. The articles “a”, “an”,and “the” as used herein in the specification and in the claims, unlessclearly indicated to the contrary, should be understood to include theplural referents. Claims or descriptions that include “or” between oneor more members of a group are considered satisfied if one, more thanone, or all of the group members are present in, employed in, orotherwise relevant to a given product or process unless indicated to thecontrary or otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention encompasses variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the claims is introduced into another claimdependent on the same base claim (or, as relevant, any other claim)unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, e.g., in Markush group orsimilar format, it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth herein. It shouldalso be understood that any embodiment of the invention, e.g., anyembodiment found within the prior art, can be explicitly excluded fromthe claims, regardless of whether the specific exclusion is recited inthe specification.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. Furthermore,where the claims recite a composition, the invention encompasses methodsof using the composition and methods of making the composition. Wherethe claims recite a composition, it should be understood that theinvention encompasses methods of using the composition and methods ofmaking the composition.

INCORPORATION OF REFERENCES

All publications and patent documents cited in this application areincorporated by reference in their entirety to the same extent as if thecontents of each individual publication or patent document wereincorporated herein.

That which is claimed is:
 1. A method comprising: detecting, using aplurality of labeled nucleic acid molecules that each comprise afragment of a cystic fibrosis transmembrane conductance regulator geneand that specifically hydbridize to a mutant but not a wild-type cysticfibrosis transmembrane conductance regulator gene, wherein the labelednucleic acid molecules each contain a cystic fibrosis transmembraneconductance regulator 2957delT mutation, and wherein the label comprisesone of a radionucleotide, a fluorophore, a chemiluminescent agent, amicroparticle, an enzyme, a colorimetric label, a magnetic label, ahapten, a molecular beacon, or an aptamer beacon, in a sample obtainedfrom a human subject, the presence of a 2957delT mutation in a cycsticfibrosis transmembrane conductance regulator (CFTR) gene or protein. 2.The method of claim 1, further comprising detecting one or more of a269C>T, 2902G>T, 3814G>A, 502G>C, 1520G>T, 511-513 dup TTA, 978A>T,843G>C, 829C>T, 4096-6C>T, 4375-7delT, 1586G>C, 875+4G>T, or 4005+3G>Tmutation of the cystic fibrosis transmembrane conductance regulator(CFTR) gene or protein using a plurality of labeled nucleic acidmolecules that each comprise a fragment of a cystic fibrosistransmembrane conductance regulator gene and that specifically hybridizeto a mutant but not a wild-type cystic fibrosis transmembraneconductance regulator gene, wherein at least one of the labeled nucleicacid molecules contains a cystic fibrosis transmembrane conductanceregulator 269C>T, 2902G>T, 3814G>A, 502G>C, 1520G>T, 511-513 dup TTA,978A>T, 843G>C, 829C>T, 4096-6C>T, 4375-7delT, 1586G>C, 875+4G>T, or4005+3G>T mutation, and wherein the label comprises one of aradionucleotide, a fluorophore, a chemiluminescent agent, amicroparticle, an enzyme, a colorimetric label, a magnetic label, ahapten, a molecular beacon, or an aptamer beacon.
 3. The method of claim1, further comprising detecting one or more of a 2711T>C, 3891G>C,2524C>T or 2894G>A mutation of the cystic fibrosis transmembraneconductance regulator (CFTR) gene or protein using a plurality oflabeled nucleic acid molecules that each comprise a fragment of a cysticfibrosis transmembrane conductance regulator gene and that specificallyhybridize to a mutant but not a wild-type cystic fibrosis transmembraneconductance regulator gene, wherein at least one of the labeled nucleicacid molecules contains a cystic fibrosis transmembrane conductanceregulator 2711T>C, 3891G>C, 2524C>T or 2894G>A mutation, and wherein thelabel comprises one of a radionuclelotide, a fluorophore, achemiluminescent agent, a microparticle, an enzyme, a colorimetriclabel, a magnetic label, a hapten, a molecular beacon, or an aptamerbeacon.
 4. The method of claim 1, further comprising detecting one ormore of a 405+10247C>T, 405+10255 del C, 1811+1643 G>T, 1812-13A>G,2752-33insA, 3849+12192G>A, 724G>A, 3899C>T, 3986C>T, 901G>A, 392T>C,3463T>C, 1757G>A, 4025G>C, 4129G>T, 663T>G, 3200T>C, 4412T>C, 620A>C,1738A>G, 3370A>C, 1129C>T, 2383C>T, 2761delTCT, 1106A>G or 622A>Gmutation of the cystic fibrosis transmembrane conductance regulator(CFTR) gene or protein using a plurality of labled nucleic acidmolecules that each comprise a fragment of a cystic fibrosistransmembrane conductance regulator gene and that specifically hybridizeto a mutant but not a wild-type cystic fibrosis transmembraneconductance regulator gene, wherein at least one of the labeled nucleicacid molecules contains a cystic fibrosis transmembrane conductanceregulator 405+10247C>T, 405+10255 del C, 1811 +1643 G>T, 1812-13A>G,2752-33insA, 3849+12192G>A, 724G>A, 3899C>T, 3986C>T, 901G>A, 392T>C,3463T>C, 1757G>A, 4025G>C, 4129G>T, 663T>G, 3200T>C, 4412T>C, 620A>C,1738A>G, 3370A>C, 1129C>T, 2383C>T, 2761delTCT, 1106A>G or 622A>Gmutation, and wherein the label comprises one of a radionucleotide, afluorophore, a chemiluminescent agent, a microparticle, an enzyme, acolorimetric label, a magnetic label, a hapten, a molecular beacon, oran aptamer beacon.
 5. The method of claim 1, further comprisingdetecting a 1824delA mutation of the cystic fibrosis transmembraneconductance regulator (CFTR) gene or protein using a plurality oflabeled nucleic acid molecules that each comprise a fragment of a cysticfibrosis transmembrane conductance regulator gene and that specificallyhybridize to a mutant but not a wild-type cystic fibrosis transmembraneconductance regulator gene, wherein the labeled nucleic acid moleculeseach contain a cystic fibrosis transmembrane conductance regulator1824delA mutation, and wherein the label comprises a radionucleotide, afluorophore, a chemiluminescent agent, a microparticle, an enzyme, acolormetric label, a magnetic label, a hapten, a molecular beacon, or anaptamer beacon.
 6. The method of claime 1, further comprising detectingone or more of a 4089insT, 4374+2T>C, 3064A>T, or 246C>G mutation of thecystic fibrosis transmembrane conductance regulator (CFTR) gene orprotein using a plurality of labeled nucleic acid molecules that eachcomprise a labeled nucleic acid molecule that comprises a fragment of acystic fibrosis transmembrane conductance regulator gene and thatspecifically hybridizes to a mutant but not a wild-type cystic fibrosistransmembrane conductance regulator gene, wherein at least one of thelabeled nucleic acid molecules contains a cystic fibrosis transmembraneconductance regulator 4089insT, 4374+2T>C, 3064A>T, or 246C>G mutation,and wherein the label comprises one of a radionucleotide, a fluorophore,a chemiluminescent agent, a microparticle, an enzyme, a colorimetriclable, a magnetic label, a hapten, a molecular beacon, or an aptamerbeacon.